Antibody Therapy for Amyloid Beta Disease

ABSTRACT

The present disclosure relates generally to a protocol to treat or prevent diseases or conditions associated with pathological forms of amyloid beta (Aβ), including Alzheimer&#39;s disease. Enabled herein is a reagent for use in the treatment and prophylaxis of Aβ-associated pathological conditions.

FILING DATA

This application is associated with and claims priority from AustralianProvisional Patent Application No. 2013902922, filed on 5 Aug. 2013,entitled “An antibody therapy for amyloid beta disease”, the entirecontents of which, are incorporated herein by reference.

BACKGROUND Field

The present disclosure relates generally to a protocol to treat orprevent diseases or conditions associated with pathological forms ofamyloid beta (Aβ), including Alzheimer's disease. Enabled herein is areagent for use in the treatment and prophylaxis and diagnosis ofAβ-associated pathological conditions.

Prior Art

Bibliographic details of the publications referred to by author in thisspecification are collected alphabetically at the end of thedescription.

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in any country.

Alzheimer's disease is a degenerative brain disorder characterizedhistologically by neuritic plaques found primarily in association withthe cortex, limbic system and basal ganglia. These neuritic plaquescomprise a cleavage product of amyloid precursor protein (APP), a type Itransmembrane glycoprotein.

The cleavage product of APP is β-amyloid peptide or Aβ. Incorrectprocessing of APP can result in pathological forms of Aβ (Tanzi et al.(1996) Neurobiol Dis 3:159-168; Hardy (1996) Ann Med 28:255-258; Schenket al. (1999) Nature 400:173-177). These pathological forms includeAβ₁₋₄₂ and Aβ₁₋₄₃ which have been detected as predominant species in theneurite plaques.

Bard et al. (2000) Nature Medicine 6:916-919 showed that peripheraladministration of antibodies directed against Aβ can reduce plaqueburden. Bard et al. (2003) Proc. Natl. Acad. Sci. USA 100:2023-2028subsequently showed that Fc-mediated phagocytosis by microglial cells ormacrophages is associated with plaque clearance. Hence, antibody therapyhas the potential to treat Alzheimer's disease.

This is supported by non-Fc-mediated mechanisms being found to beassociated with Aβ clearance in immunotherapy (Bacskai et al. (2002). J.Neurosci. 22:7873-7878; Das et al. (2003) J. Neurosci. 23:8532-9538).

One attempt at immunotherapy was the humanized antibody, Bapineuzumab orBapi, developed by Pfizer and Johnson & Johnson. Bapi targets neurotoxicAβ (Salloway et al. (2009) Neurology 73:2061-2070) at the extremeN-terminus in a helical conformation (Miles et al. (2013) Sci Rep 3:1-8)[doi:10.1038/srep01302]. Such Aβ forms with the N-terminal truncationscomprise approximately 60% of Aβ deposits in the Alzheimer's diseasedbrains. Bapi was, however, shown to be toxic at higher doses.

Another antibody is Solanezumab (Eli Lilly). This antibody targetsmonomeric Aβ in the mid-region of the peptide and was partiallyefficacious in mild cases of Alzheimer's disease (Hardy (2014) N Engl JMed 370(4):377-378).

However, it appears that Solanezumab may be ineffective at reversing thesymptoms in the later stages of Alzheimer's disease (Panza et al. (2014)Expert Opin Biol Ther: 1-12 PMID 24981190).

Crenezumab (Genentech Inc.) also targets the mid-region of Aβ. Itappears to stimulate microglia to a level sufficient to clear Aβ butwithout inducing an inflammatory response (Adolfsson et al. (2012) J.Neuroscience 32(28):9677-9689).

Ponezumab (Pfizer) targets the C-terminal end of Aβ₁₋₄₀ but is unable tobind to elongated forms such as Aβ₁₋₄₂ and Aβ₁₋₄₃. Ponezumab was notefficacious in reducing biomarkers of Alzheimer's disease or cognitivedecline (Liu et al. (2014) Mol Neurobiol:PMID 24733588). Anothermonoclonal antibody which has the same binding specificity as Ponezumabis referred to as Mab 2286 (Rosenthal et al. USSN 2011/038861,2004/0146512 and 2007/0160616). Mab 2286 also does not bind to Aβ₁₋₄₂and Aβ₁₋₄₃.

There is a need to further develop an immunotherapeutic approach to thetreatment of disease conditions associated with toxic Aβ forms and theirdiagnosis.

SUMMARY

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2),etc. A summary of the sequence identifiers is provided in Table 1. Asequence listing is provided after the claims.

The present disclosure teaches the generation of an antibody andderivatives thereof which target Aβ₁₋₄₀ and its C-terminally extendedforms including Aβ₁₋₄₂ and Aβ₁₋₄₃. The inventors analyzed the molecularbasis of antibody engagement with Aβ. The 3D structure of a Fab bindingfragment of the variable domain of Mab 2286 (referred to herein as “Fab2286”) was determined to near atomic resolution. Fab 2286 is a chimeracomprising the mouse variable region of Mab 2286 transplanted onto ahuman scaffold. Fab 2286 comprises V_(H) and V_(L) from Mab 2286 andC_(H) and C_(L) from a human template. This antibody exhibits similarspecificity as Ponezumab to the C-terminus of Aβ₁₋₄₀ and does not reactwith Aβ₁₋₄₂ or Aβ₁₋₄₃ or other elongated forms such as Aβ₃₋₄₂, Aβ₄₋₄₂,Aβ_(pyroGln3-42) and Aβ_(pyroGlu11-42). The analysis of chimeric Fab2286 revealed a hydrophobic cavity in the Fab 2286 surface suitable forbinding amyloid beta peptide. The amino acid sequences of Ponezumab andFab 2286 show little similarity in the complementarity determiningregions, but comparison of the 3D structures (Protein Data Bank entries3U0T and 3U0W, respectively) revealed some potential spacialsimilarities for ligand engagement. Structural analysis of thePonezumab-Aβ complex revealed a buried Aβ terminal carboxyl locationthat excluded cross reactivity with C-terminally elongated Aβ species. Amodification is prepared of the proposed Aβ₁₋₄₀ C-terminus binding siteof Fab 2286 at the glutamic acid (E; Glu) at residue 50 in the heavyvariable chain of Fab 2286 [Glu50]. The substitution to another aminoacid (Glu500Xaa), wherein Xaa is not glutamic acid, substantiallyimproves affinity for the Aβ₁₋₄₀ ligand and enables binding to theC-terminally elongated Aβ species. In an embodiment, Xaa is selectedfrom the list consisting of alanine, arginine, asparagine, asparticacid, cysteine, glutamine, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine, pyrrolysine and selenocysteine. In anembodiment, Xaa is a basic amino acid such as arginine, lysine orhistidine. In an embodiment, Xaa is alanine. In a particular embodiment,Xaa is arginine.

Accordingly, enabled herein is an antibody that specifically binds tothe C-terminal end portion of Aβ₁₋₄₀ and to C-terminally extended toxicforms thereof. In an embodiment, the antibody is a derivative of Fab2286 and is referred to herein as a “Fab 2286-like antibody”. Fab 2286comprises the antigen binding fragment of Mab 2286 grafted onto a humanscaffold constant region. In an embodiment, the instant disclosureteaches a Fab 2286-like antibody comprising an amino acid substitutionat glutamic acid (E) in its heavy chain in the amino acid sequence WIGEto generate the sequence WIGX. In an embodiment, the substitution is toan amino acid selected from the list consisting of alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, valine, pyrrolysine and selenocysteine.In an embodiment, the substitution is to a basic amino acid such asarginine, lysine or histidine. In an embodiment, the substitution is toan alanine. In an embodiment, the substitution is to an arginine. TheFab 2286-like antibody may have one or more other mutations. The Fab2286-like antibody may be conjugated to any light and heavy chainconstant region and be subject to mammalianization such as humanization(or deimmunization). In terms of testing the antibody in an animalmodel, the antibody may, for example, be subject to murinization.

Further enabled herein is a method for treating a disease or conditioncharacterized by the presence of aberrant or toxic forms of Aβ includingC-terminally elongated forms (e.g. Aβ₁₋₄₂, Aβ₁₋₄₃. Aβ₃₋₄₂, Aβ₃₋₄₂,Aβ₄₋₄₂, Aβ_(pyroGlu3-42) and Aβ_(pyroGlu11-42)) as well as covalentlycross-linked pathogenic forms of Aβ multimers. The method comprises theadministration of a Fab 2286-like antibody comprising a mutation in itsheavy chain variable region which enables it to bind to C-terminallyelongated pathogenic forms of Aβ. The mutation is a Glu50Xaasubstitution in SEQ ID NO: 1. An example is a Glu500Arg substitution asdepicted in SEQ ID NO:2. Other modified forms of the heavy chain whichincludes the Glu50Arg substitution are provided in SEQ ID NOs:6 and 8.As indicated above, however, the glutamic acid is changed to an aminoacid selected from the list consisting of alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine, pyrrolysine and selenocysteine. In anembodiment, the amino acid is a basic amino acid such as arginine,lysine or histidine. In an embodiment, the amino acid is alanine. In anembodiment, the amino acid is arginine.

Diseases and conditions contemplated herein include Alzheimer's disease,Down's syndrome, cognitive impairment or memory loss, Parkinson'sdisease multi-infarct dementia, cerebral amyloid angiopathy, glaucomaand a vascular disorder caused by pathogenic Aβ peptide in blood vessels(e.g. stroke and hereditary cerebral hemorrhage with amyloidosis-Dutchtype [HCHWA-D]). The subject antibody may also be used to capture and/ordetect toxic forms of Aβ associated with the above-listed conditions. Interms of diagnosis, another condition contemplated herein ispre-eclampsia. Whilst pre-eclampsia and a neurological condition such asAlzheimer's disease share no clinical features in common, thepre-eclampsia susceptibility gene, STOX-1, is abundantly expressed inthe brain and transactivates LRRTM3 in neural cells which promoteselevated Aβ processing. Hence, the presence of toxic forms of Aβ inurine is potentially indicative of pre-eclampsia. The Fab 2286-likeantibody may act to promote microglial-mediated removal of pathogenic Aβspecies in the brain and/or through the “peripheral sink” route toinduce an equilibrium shift to remove Aβ from the brain to the bloodstream where it is removed by various immune mechanisms.

The treatment may also include ameliorating symptoms or delaying onsetof symptoms of an Aβ pathology such as Alzheimer's disease.

Another aspect contemplated herein is a method of detecting a toxic formof Aβ in a sample from a subject, the method comprising identifyingbinding between the Aβ form and Fab 2286-like antibody. The antibody mayalso be used to capture an Aβ form which is then detected by, forexample, another antibody specific for an epitope on Aβ or by ananti-immunoglobulin antibody which binds to the Fab 2286-like antibody.

Diagnostic applications extend to Alzheimer's disease, Down's syndrome,cognitive impairment or memory loss, Parkinson's disease multi-infarctdementia, cerebral amyloid angiopathy, glaucoma and a vascular disordercaused by pathogenic Aβ peptide in blood vessels (e.g. stroke andhereditary cerebral hemorrhage with amyloidosis-Dutch type [HCHWA-D])and pre-eclampsia. Whilst a blood-based test is proposed for mostconditions, for pre-eclampsia, a urine-based test is proposed. Cerebralspinal fluid may also be tested. In an embodiment, taught herein is adipstick comprising Fab 2286-like antibody immobilized thereon for usein detecting toxic forms of Aβ in a biological sample. In an embodiment,the sample is urine and the condition diagnosed is pre-eclampsia.

TABLE 1 Summary of sequence identifiers SEQUENCE ID NO: DESCRIPTION 1Amino acid sequence of heavy chain variable region of Fab 2286 antibody2 Amino acid sequence of heavy chain of Fab 2286-like antibodycomprising Glu50Arg substitution 3 Amino acid sequence of light chain(hkappa) of Fab 2286 and Fab 2286-like antibody) 4 Amino acid sequenceof Aβ₁₋₂₈ 5 Aniino acid sequence of Aβ₁₋₄₀ 6 Amino acid sequence ofAβ₁₋₄₂ 7 Amino acid sequence of Aβ₁₋₄₃ 8 Amino acid sequence of Aβ₃₅₋₄₀9 Amino acid sequence, of murinized Fab 2286-like antibody heavy chainconjugated to human gamma-1 immunoglobulin (hG1) heavy constant regionwith Glu50Arg substitution 10 Amino acid sequence of chimeric Fab 2286antibody heavy chain variable region 11 Amino acid sequence of chimericFab 2286-like antibody heavy chain variable region 12 Nucleotidesequence of Mab 2286 heavy chain (variable domain and constant domain 1[CH1]) 13 Amino acid sequence of Mab 2286 heavy chain (variable domainand constant domain 1 [CH1]) 14 Nucleotide sequence of Mab 2286 lightchain 15 Amino acid sequence of Mab 2286 light chain 16 Nucleotidesequence of synthetic DNA construct encoding Fc portion of murine gammaheavy chain (GenBank: AAA75163.1) of Mab 2286 17 Amino acid sequence ofFc portion of murine gamma heavy chain (GenBank: AAA75163.1) of Mab 228618 Nucleotide sequence of murine heavy B encoding Glu to Argsubstitution 19 Amino acid sequence of murine heavy B with Glu to Argsubstitution 20 Nucleotide sequence of murine light chain of Fab2286-like antibody 21 Amino acid sequence of murine light chain of Fab2286-like antibody

Amino acid abbreviations used herein are defined in Table 2.

TABLE 2 Amino acid three and single letter abbreviations Three-letterOne-letter Amino Acid Abbreviation Symbol Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln QGhitamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile ILeucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F ProlinePro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr YValine Val V Pyrrolysine Pyl O Selenocysteine Sec U Any residue Xaa X

BRIEF DESCRIPTION OF THE FIGURES

Some figures contain color representations or entities. Colorphotographs are available from the Patentee upon request or from anappropriate Patent Office. A fee may be imposed if obtained from aPatent Office.

FIGS. 1A through C are photographical representations of SDS-PAGEanalysis of synthetic Aβ preparations. Detected by (A) Coomassie stain,(B) anti-Aβ40 C-terminus specific Mab 2286 Western Blot and (C)anti-Aβ₂₋₇ specific Mab WO2 Western Blot. In each panel, lanesrepresent: (1) Aβ₁₋₄₀ aged for 21 days at room temperature in PBS; (2)freshly prepared, from 2,2,2-trifluoroethanol (TFE)-treated andlyophilized stock, Aβ₁₋₄₀; (3) Aβ₁₋₄₂ aged for 21 days at roomtemperature in phosphile buffered saline (PBS); (4) freshly prepared,from TFE-treated and lyophilized stock, Aβ₁₋₄₂; (5) 1:1 mixture ofAβ₁₋₄₀ and Aβ₁₋₄₂ aged overnight at room temperature in PBS; (6) Aβ₁₋₂₈freshly prepared, from TFE-treated and lyophilized stock.

FIG. 2 is a graphical representation of the binding characteristics ofFab 2286 (Aβ Fab wt) and the Fab 2286-like antibody (Aβ Fab mutant) intissue from a familial Alzheimer's diseased brain and against syntheticpeptide Aβ₁₋₄₂.

FIG. 3 is a graphical representation of the binding characteristics ofFab 2286 (Aβ Fab wt) and the Fab 2286-like antibody (Aβ Fab mutant) intissue from a sporadic Alzheimer's diseased brain and against syntheticpeptide Aβ₁₋₄₂.

DETAILED DESCRIPTION

Throughout this specification, unless the context requires otherwise,the word “comprise” or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror method step or group of elements or integers or method steps but notthe exclusion of any element or integer or method step or group ofelements or integers or method steps.

As used in the subject specification, the singular forms “a”, “an” and“the” include plural aspects unless the context clearly dictatesotherwise. Thus, for example, reference to “a mutation” includes asingle mutation, as well as two or more mutations; reference to “anantibody” includes a single antibody, as well as two or more antibodies;reference to “the disclosure” includes a single reference to “thedisclosure” and includes single and multiple aspects taught by thedisclosure; and so forth. Aspects taught and enabled herein areencompassed by the term “invention”. All such aspects are enabled withinthe width of the present invention.

Disclosed herein is an antibody which binds to Aβ₁₋₄₀ (“Aβ₄₀”) as wellas C-terminally elongated pathogenic forms of Aβ, including Aβ₁₋₄₂(“Aβ₄₂”) and Aβ₁₋₄₃ (“Aβ₄₃”) as well as covalently linked multimersthereof. A “multimer” includes an oligomer. The antibody comprises amature heavy chain having the amino acid sequence as set forth in SEQ IDNO: 1 with the proviso that in the sequence WIGE, the glutamic acid (E)is substituted for another amino acid residue, i.e. Glu50Xaa. In anembodiment, Xaa is selected from the list consisting of alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, valine, pyrrolysineand selenocysteine. In an embodiment, Xaa is a basic amino acid such asarginine, lysine or histidine. In an embodiment, Xaa is alanine. In anembodiment, Xaa is arginine such as depicted in SEQ ID NO:2. Other formsare provided in SEQ ID NOs:9 and 11. The amino acid sequence of itslight chain is set forth in SEQ ID NO:3. The mature heavy chain isderived from an antigen-binding fragment of Mab 2286 (a Fab 2286fragment) and the antibody of the present disclosure is referred to asan “Fab 2286-like antibody”. It is also referred to as “Aβ Fab mutant”.The Mab 2286 precursor antigen binding fragment is referred to as “Fab2286” or “Aβ Fab wt”. The Fab 2286-like antibody differs from Ponezumaband Mab 2286 by its ability to bind to C-terminally elongated Aβspecies. These include Aβ₁₋₄₂ and Aβ₁₋₄₃ as well as Aβ₃₋₄₂, Aβ₄₋₄₂,Aβ_(pyroGlu3-42) and Aβ_(pyroGlu11-42). The Fab 2286-like antibody mayalso comprise one or more additional amino acid substitutions, additionsand/or deletions. In addition, the Fab 2286-like antibody may beconjugated to any heavy and light constant regions. For example, in anembodiment, the Fab 2286-like antibody is conjugated to human kappa-1immunoglobulin (hkappa) light chain and human gamma-1 immunoglobulin(hG1) heavy chain constant regions. In one example, the heavy chainvariable and constant regions (V_(H)+C_(H)) have an amino acid sequenceset forth in SEQ ID NO:9. Furthermore, the antibody may be mammalianizedso that it can be administered to a particular mammal such as a mouse,rat, pig, sheep or monkey. In an embodiment, the mammal is a human andthe antibody is humanized or deimmunized. In terms of testing theantibody in an animal model such as a mouse model, it may also besubject to murinization.

Accordingly, enabled herein is an isolated Fab 2286-like antibody whichbinds to Aβ₁₋₄₀ and a C-terminally elongated form thereof, the antibodycomprising a modified Fab 2286 wherein a mature heavy chain variableregion comprises an amino acid sequence as set forth in SEQ ID NO:1 withthe proviso that the amino acid residue at position 50 is not glutamicacid, wherein the numbering of the amino acid sequence WIGE in Fab 2286(SEQ ID NO:1) represents residues 47 to 50 or comprising one or moreother amino acid substitutions, additions and/or deletions to the aminoacid sequence of SEQ ID NO:1.

Enabled herein is an isolated antibody comprising a mature heavy chainas set forth in SEQ ID NO:2 or one or more amino acid substitutions,additions and/or deletions to the amino acid sequence set forth in SEQID NO:2 provided that the amino acid residue at position 50 is arginine(R) [50 Arg]. In an embodiment, the amino acid sequence comprises atleast 90% similarity to SEQ ID NO:2 provided it still contains theGlu50Arg substitution.

Enabled herein is an isolated antibody comprising a mature heavy chainas set forth in SEQ ID NO:9 or one or more amino acid substitutions,additions and/or deletions to the amino acid sequence set forth in SEQID NO:9 provided that the amino acid residue at position 50 is arginine(R) [50 Arg]. In an embodiment, the amino acid sequence comprises atleast 90% similarity to SEQ ID NO:9 provided it still contains theGlu50Arg substitution.

Enabled herein is an isolated antibody comprising a mature heavy chainas set forth in SEQ ID NO:11 or one or more amino acid substitutions,additions and/or deletions to the amino acid sequence set forth in SEQID NO:11 provided that the amino acid residue at position 50 is arginine(R) [50 Arg]. In an embodiment, the amino acid sequence comprises atleast 90% similarity to SEQ ID NO:1 provided it contains the Glu50Argsubstitution.

To take into account variations in the heavy chain, in the amino acidsequence WIGE of SEQ ID NO:1 (mature heavy chain variable region of Fab2286), the E is at position 50 and in the Fab 2286-like antibody, thesequence is WIGR (see SEQ ID NO:2, 9 and 11). This is also referred toherein as a Glu50Arg substitution of the mature heavy chain variableregion of Fab 2286. However, the present invention extends to asubstitution of E at position 50 to any amino acid residue such asalanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine,pyrrolysine or selenocysteine. In an embodiment, the substitution is toa basic amino acid residue such as arginine, lysine or histidine. In anembodiment, the substitution is to an alanine. In an embodiment, thesubstitution is to an arginine. In an embodiment, the present inventionextends to a Fab 2286-like antibody comprising a heavy chain variableregion as set forth in SEQ ID NO: 1 with the exception that in thesequence WIGE at amino acids 47 to 50, the sequence is selected fromWIGA, WIGR, WIGN, WIGD, WIGC, WIGQ, WJGG, WIGH, WIGI, WIGL, WIGK, WIGM,WIGF, WIGP, WIGS, WIGT, WIGW, WIGY, WIGV, WIGO and WIGU. In anembodiment, the sequence is WIGR, WIGK, WIGH, WIGA or WIGM. In anembodiment, the seuqnece is WIGR. Reference to “at least 90%” amino acidsequence similarity means 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%similarity. By “similarity” includes “identity”.

In a related embodiment, enabled herein is an isolated antibody whichbinds to Aβ₁₋₄₀ or C-terminally elongated forms thereof or covalentlylinked multimers thereof, the antibody comprising a modified form of Fab2286 wherein a mature heavy chain variable region comprises a modifiedamino acid sequence set forth in SEQ ID NO:1 with the proviso that theamino acid residue at position 50 is not glutamic acid (E), wherein thenumbering of the amino acid sequence WIGE in Fab 2286 (SEQ ID NO:1)represents amino acid residues 47 to 50 or comprising one or more aminoacid substitutions, additions and/or deletions to the amino acidsequence of SEQ ID NO: 1.

In an embodiment, the amino acid residue at position 50 is selected fromthe list consisting of alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, valine, pyrrolysine and selenocysteine. In an embodiment, theamino acid residue at position 50 is a basic amino acid such asarginine, lysine or histidine. In an embodiment, the amino acid residueat position 50 is alanine. In an embodiment, the amino acid reisdue atposition 50 is arginine.

The present disclosure is instructional on a Fab 2286-like antibodycomprising a mature heavy chain variable region comprising the aminoacid sequence set forth in SEQ ID NO:2 wherein the Fab 2286-likeantibody binds to Aβ₁₋₄₀ and to C-terminally elongated forms thereof.

Other examples of the heavy chain variable regions are found in SEQ IDNOs:9 and 11.

Reference to “C-terminally elongated forms” of Aβ₁₋₄₀ includes Aβ₁₋₄₂,Aβ₁₋₄₃, Aβ₃₋₄₂, Aβ₄₋₄₂, Aβ_(pyroGlu3-42) and Aβ_(pyroGlu11-42).

Enabled herein is an isolated polynucleotide encoding an amino acidsequence of a mature heavy chain variable region of an antibody whichbinds to Aβ₁₋₄₀ and C-terminally elongated forms thereof or covalentlylinked multimers thereof, the antibody comprising a modified form of Fab2286 wherein a mature heavy chain variable region comprises the aminoacid sequence set forth in SEQ ID NO: 1 wherein the amino acid atposition 50 is not glutamic acid or comprising one or more other aminoacid substitutions, additions and/or deletions to the amino acidsequence of SEQ ID NO: 1. The numbering of the amino acid sequence WIGEin Fab 2286 (SEQ ID NO:1) represents amino acid residues 47 to 50. In anembodiment, the amino acid at position 50 is selected from the listconstiting of alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine,pyrrolysine and selenocysteine. In an embodiment, the amino acid atposition 50 is a basic amino acid such as arginine, lysine or histidine.In an embodiment, the amino acid at position 50 is alanine. In anembodiment, the amino acid residue at position 50 is arginine. Hence, inan embodiment, the polynucleotide encodes SEQ ID NO:2. Cells and vectorscomprising the polynucleotide are also enabled herein. Polynucleotideencoding SEQ ID NOs:9 and 11 are also contemplated herein as well aspolynucleotide sequence which have at least 90% identity to thenucleotide sequence encoding each of SEQ ID NO:2, 9 or 11 or a nucleicacid capable of hybridizing under medium or high stringency conditionsto the nucleotide sequence encoding SEQ ID NO:2, 9 or 11 provided thenucleotide sequence encodes Glu50Arg substitution. In an embodiment, thenucleotide sequence is SEQ ID NO:18 or a nucleotide having at least 90%identity to SEQ ID NO:18 or a nucleotide sequence which hybridizes tothe complement of SEQ ID NO: 18 under medium or high stringencyconditions. In relation to a nucleotide sequence, “at least 90%” means90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

The term “similarity” as used herein includes exact identity betweencompared sequences at the nucleotide or amino acid level. Where there isnon-identity at the nucleotide level, “similarity” includes differencesbetween sequences which result in different amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. Where there is non-identity atthe amino acid level, “similarity” includes amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. In an embodiment, nucleotideand sequence comparisons are made at the level of identity rather thansimilarity.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence”,“comparison window”, “sequence similarity”, “sequence identity”,“percentage of sequence similarity”, “percentage of sequence identity”,“substantially similar” and “substantial identity”. A “referencesequence” is at least 12 but frequently 15 to 18 and often at least 25or above, such as 30 monomer units, inclusive of nucleotides and aminoacid residues, in length. Because two polynucleotides may each comprise(1) a sequence (i.e. only a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2) asequence that is divergent between the two polynucleotides, sequencecomparisons between two (or more) polynucleotides are typicallyperformed by comparing sequences of the two polynucleotides over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window” refers to a conceptual segment oftypically 12 contiguous residues that is compared to a referencesequence. The comparison window may comprise additions or deletions(i.e. gaps) of about 20% or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by computerized implementations ofalgorithms (e.g. GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package Release 7.0, Genetics Computer Group, 575Science Drive Madison, Wis., USA) or by inspection and the bestalignment (i.e. resulting in the highest percentage homology over thecomparison window) generated by any of the various methods selected.Reference also may be made to the BLAST family of programs as forexample disclosed by Altschul et al. (1997) Nucl. Acids. Res. 25:3389. Adetailed discussion of sequence analysis can be found in Unit 19.3 ofAusubel et al. (In: Current Protocols in Molecular Biology, John Wiley &Sons Inc. 1994-1998).

The terms “sequence similarity” and “sequence identity” as used hereinrefers to the extent that sequences are identical or functionally orstructurally similar on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity”, for example, is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala,Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp,Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. For the purposes of the present invention, “sequenceidentity” will be understood to mean the “match percentage” calculatedby the DNASIS computer program (Version 2.5 for windows; available fromHitachi Software engineering Co., Ltd., South San Francisco, Calif.,USA) using standard defaults as used in the reference manualaccompanying the software. Similar comments apply in relation tosequence similarity.

Reference to medium stringency includes and encompasses from at leastabout 16% v/v to at least about 30%0/v/v formamide and from at leastabout 0.5 M to at least about 0.9 M salt for hybridization, and at leastabout 0.5 M to at least about 0.9 M salt for washing condition.Reference to high stringency includes and encompasses from at leastabout 31% v/v to at least about 50% v/v formamide and from at leastabout 0.01 M to at least about 0.15 M salt for hybridization, and atleast about 0.01 M to at least about 0.15 M salt for washing conditions.In general, washing is carried out T_(m)=69.3+0.41 (G+C) % (Marmur andDoty (1962) J. Mol. Biol. 5:109). However, the T_(m) of a duplex DNAdecreases by 1° C. with every increase of 1% in the number of mismatchbase pairs (Bonner and Laskey (1974) Eur. J. Biochem. 46:83). Formamideis optional in these hybridization conditions. Accordingly, particularlypreferred levels of stringency are defined as follows: low stringency is6×SSC buffer, 0.1% w/v SDS at 25°-42° C.; a moderate stringency is 2×SSCbuffer, 0.1% w/v SDS at a temperature in the range 20° C. to 65° C.;high stringency is 0.1×SSC buffer, 0.1% w/v SDS at a temperature of atleast 65° C.

The Fab 2286-like antibody disclosed herein is useful in the treatmentof a subject with a disease or condition associated with orcharacterized by the presence of pathogenic or toxic forms of Aβspecies. The C-terminally elongated Aβ species may also be in the formof cerebral deposits. Such diseases or conditions include Alzheimer'sdisease, Down's syndrome, cognitive impairment or memory loss,Parkinson's disease, multi-infarct dementia, cerebral amyloidangiopathy, glaucoma and a vascular disorder caused by pathogenic Aβpeptide in blood vessels (e.g. stroke and hereditary cerebral hemorrhagewith amyloidosis-Dutch type [HCHWA-D]). Generally, the subject is ahuman although for testing purposes, non-human mammalian subjects may betested. In an example, a humanized antibody is used in humans and amurinize form is used for mice or rats. In an embodiment, the subject isa companion animal such as a dog or cat (using caninized and felinizedantibodies, respectively). See, for example, SEQ ID NOs:9 and 11.

Hence, the instant disclosure teaches a method for the treatment orprophylaxis of a condition associated with or characterized by thepresence of a pathogenic form of Aβ comprising a C-terminally elongatedspecies of Aβ, the method comprising administering to a subject in needof treatment an effective amount of an antibody comprising a matureheavy chain having a modified amino acid sequence set forth in SEQ IDNO: 1 with the proviso that the amino acid residue at position 50 is notglutamic acid, using a numbering system where the amino acid sequenceWIGE in Fab 2286 (SEQ ID NO:1) is at positions 47 to 50 or an amino acidsequence with one or more other amino acid substitutions, additionsand/or deletions thereto. In an embodiment, the amino acid at position50 is selected from the list consisting of alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, valine, pyrrolysine and selenocysteine.In an embodiment, the amino acid at position 50 is a basic amino acidsuch as arginine, lysine or histidine. In an embodiment, the amino acidat position 50 is alanine. In an embodiment, the amino acid at position50 is arginine.

In an embodiment, the instant disclosure teaches a method for thetreatment or prophylaxis of a condition associated with or characterizedby the presence of a pathogenic form of Aβ comprising a C-terminallyelongated species of Aβ, the method comprising administering to asubject in need of treatment an effective amount of an antibodycomprising a mature heavy chain having the amino acid sequence set forthin SEQ ID NO:2 or an amino acid sequence with one or more amino acidsubstitutions, additions and/or deletions thereto with the proviso thatthe amino acid residue at position 50 is R, using a numbering systemwhere the amino acid sequence WIGE in Fab 2286 (SEQ ID NO: 1) is atpositions 47 to 50.

The effective amount of antibody includes an amount sufficient formicroglia to be stimulated to facilitate removal of pathogenic forms ofAβ to which the antibody binds. Generally, the antibody does notsubstantially provoke an inflammatory response, the Fab 2286-likeantibody promotes clearance of pathogenic Aβ species. This can be viamicroglial removal of Aβ in the brain or via a shift in the equilibriumof Aβ from the brain to the circulatory blood system (“peripheralsink”).

Enabled herein is a method of treating or ameliorating symptoms of aneurological condition selected from Alzheimer's disease, Down'ssyndrome, cognitive impairment or memory loss, Parkinson's disease,multi-infarct dementia, cerebral amyloid angiopathy, glaucoma, strokeand HCHWA-D or other adverse event involving amyloidosis in a subject,the method comprising administering to the subject, an effective amountof a Fab 2286-like antibody comprising a mature heavy chain variableregion having a modified amino acid sequence set forth in SEQ ID NO: 1with the proviso that the amino acid residue at position 50 is notglutamic acid (E) using a numbering system where the amino acid sequenceWIGE of Fab 2286 (SEQ ID NO: 1) is at positions 47 to 50 or having oneor more other amino acid substitutions, additions and/or deletionsthereto. In an embodiment, the amino acid substitution may also bereferred to as Glu50Xaa in the heavy chain variable region of Fab 2286.In an embodiment, Xaa is selected from the list consisting of alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, valine, pyrrolysineand selenocysteine. In an embodiment, Xaa is a basic amino acid residuesuch as arginine, lysine or histidine. In an embodiment, Xaa is alanine.In a particular embodiment, Xaa is arginine (i.e. the substitution isGlu50Arg).

The term “Fab 2286-like antibody” means that the Fab 2286 antibody hasundergone a modification to the mature heavy chain variable region tosubstitute the E in the amino acid sequence WIGE at positions 47 to 50to an R (i.e. WIGR) [Glu500Arg] or another amino acid [Glu500Xaa],wherein Xaa is not glutamic acid. One or more other amino acidsubstitutions, additions and/or deletions may also be made to the matureheavy chain or a light chain. The Fab 2286-like antibody, however, bindsto A β₁₋₄₀ and C-terminally elongated forms thereof such as A β₁₋₄₂ andA β₁₋₄₃. In addition, the Fab 2286-like antibody may be conjugated toany light and heavy chain constant region scaffold. The original 2286antibody (V_(L)+C_(L); V_(H)+C_(H)) is referred to as Mab 2286. In anembodiment, the Fab 2286-like antibody is conjugated to heavy and lightchain constant regions of any antibody.

The antibody may also bind to covalently bound multimers of these Aβspecies.

In an embodiment, the present disclosure teaches a method for thetreatment or prophylaxis of Alzheimer's disease in a subject, the methodcomprising administering to the subject an effective amount of an Fab2286-like antibody having a modification to its mature heavy chain tosubstitute an E in the amino acid sequence WIGE to X (WIGX), wherein Xis any amino acid except glutamic acid. In an embodiment, X is R (WIGR).

One or more other amino acid substitutions, additions and/or deletionsmay also be made to the heavy or light chain. In one embodiment, theV_(L)+C_(L) comprises an amino acid sequence as set forth in SEQ IDNO:9.

The inventors analyzed the molecular basis of antibody engagement of Aβepitopes. The 3D Fab structure (Fab 2286) was determined to near atomicresolution of the variable domain of Mab 2286. Fab 2286 is a chimeracomprising the mouse variable region of Mab 2286 transplanted onto ahuman scaffold. Fab 2286 is V_(H) and V_(L) from Mab 2286 and C_(H) andC_(L) from a human template. The analysis revealed a hydrophobic cavityin the Fab 2286 binding site in which the C-terminal region of A β₁₋₄₀was modeled. Data further revealed a buried terminal carboxyl locationthat excluded cross reactivity with C-terminally elongated Aβ species.Modification of the glutamic acid (E) at residue 50 by the substitutionto another amino acid residue in the mature heavy chain variable regionenables binding to the C-terminally elongated Aβ species.

Reference to an “antibody” includes an immunoglobulin molecule whichbinds to a specific target. In the case of the Fab 2286-like antibody ofthe instant disclosure, the specific target is the C-terminal end of Aβ₁₋₄₀ or C-terminally elongated Aβ species such as Aβ₁₋₄₂ and A β₁₋₄₃ aswell as Aβ₃₋₄₂, Aβ₄₋₄₂, A β_(pyroGlu3-43) and A β_(pyroGlu11-42). Theimmunoglobulin binds to the C-terminal end of the elongated pathogenicforms of Aβ. Further covered herein are fragments of antibodies such asFab, Fab′, F(ab′)₂ and Fv fragments, single chain (ScFv) forms, mutants,fusion proteins comprising a portion of the Fab 2286-like antibody andsynthetically modified derivatives. In an embodiment, the Fab 2286-likeantibody is conjugated to hkappa-1 light chain and hG1 heavy chainconstant regions.

As used herein, an “effective dosage” or “effective amount” of the Fab2286-like antibody or a pharmaceutical composition comprising same is anamount sufficient to effect beneficial or desired results. Forprophylactic use, beneficial or desired results include results such aseliminating or reducing the risk, lessening the severity, or delayingthe outset of the disease, including biochemical, histological and/orbehavioral symptoms of the disease, its complications and intermediatepathological phenotypes presenting during development of the disease.Such a disease is a disease or condition associated with pathogenicforms of Aβ. Examples include Alzheimer's disease, Down's syndrome,cognitive impairment or memory loss, Parkinson's disease, multi-infarctdementia, cerebral amyloid angiopathy, glaucoma and a vascular disordercaused by pathogenic Aβ peptide in blood vessels (e.g. stroke andhereditary cerebral hemorrhage with amyloidosis-Dutch type [HCHWA-D]).For therapeutic use, beneficial or desired results include clinicalresults such as inhibiting, suppressing or reducing the formation ofamyloid plaques, reducing, removing, clearing amyloid plaques, improvingcognition, reversing or slowing cognitive decline, sequestering orincreasing soluble Aβ peptide circulating in biological fluids,decreasing one or more symptoms resulting from the disease (biochemical,histological and/or behavioral), including its complications andintermediate pathological phenotypes presenting during development ofthe disease, increasing the quality of life of those suffering from thedisease, decreasing the dose of other medications required to treat thedisease, enhancing effect of another medication, delaying theprogression of the disease, and/or prolonging survival of patients. Aneffective dosage can be administered in one or more administrations. Forpurposes of the present disclosure, an effective dosage of antibody, orpharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective dosage of the Fab2286-like antibody or pharmaceutical composition comprising same may ormay not be achieved in conjunction with another drug, compound, orpharmaceutical composition. Thus, an “effective dosage” may beconsidered in the context of administering one or more therapeuticagents, and a single agent may be considered to be given in an effectiveamount if, in conjunction with one or more other agents, a desirableresult may be or is achieved.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including clinical results. For purposesof this disclosure, beneficial or desired clinical results include, butare not limited to, one or more of the following: inhibiting,suppressing or reducing the formation of amyloid plaques, reducing,removing, or clearing amyloid plaques, improving cognition, reversing orslowing cognitive decline, sequestering soluble Aβ peptide circulatingin biological fluids, reducing Aβ peptide (including soluble, oligomericand deposited) in a tissue (such as brain), inhibiting, slowing and/orreducing accumulation of Aβ peptide in the brain, inhibiting, slowingand/or reducing toxic effects of Aβ peptide in a tissue (such as brain),decreasing symptoms resulting from the disease, increasing the qualityof life of those suffering from the disease, decreasing the dose ofother medications required to treat the disease, delaying theprogression of the disease, and/or prolonging survival of patients.Reference to disease includes Alzheimer's disease, Down's syndrome,cognitive impairment or memory loss, Parkinson's disease, multi-infarctdementia, cerebral amyloid angiopathy, glaucoma and a vascular disordercaused by pathogenic Aβ peptide in blood vessels (e.g. stroke andhereditary cerebral hemorrhage with amyloidosis-Dutch type [HCHWA-D]).

As used herein, “delaying” development of Alzheimer's disease means todefer, hinder, slow, retard, stabilize, and/or postpone development ofthe disease or its symptoms. This delay can be of varying lengths oftime, depending on the history of the disease and/or individual beingtreated. As is evident to one skilled in the art, a sufficient orsignificant delay can, in effect, encompass prevention, in that theindividual does not develop the disease. A method that “delays”development of Alzheimer's disease is a method that reduces probabilityof disease development in a given time frame and/or reduces extent ofthe disease in a given time frame, when compared to not using themethod. Such comparisons are typically based on clinical studies, usinga statistically significant number of subjects.

“Development” of Alzheimer's disease means the onset and/or progressionof Alzheimer's disease within an individual. Alzheimer's diseasedevelopment can be detectable using standard clinical techniques.However, development also refers to disease progression that may beinitially undetectable. For purposes of this disclosure, progressionrefers to the biological course of the disease state, in this case, asdetermined by a standard neurological examination, patient interview, ormay be determined by more specialized testing such as the detection ormonitoring of biological markers of the disease. A variety of thesediagnostic tests include, but not limited to, neuroimaging, detectingalterations of levels of specific proteins in the serum or cerebrospinalfluid (e.g. amyloid peptides and Tau), computerized tomography (CT), andmagnetic resonance imaging (MRI). “Development” includes occurrence,recurrence, and onset. As used herein “onset” or “occurrence” ofAlzheimer's disease includes initial onset and and/or recurrence.

As used herein, administration “in conjunction” includes simultaneousadministration and/or administration at different times. Administrationin conjunction also encompasses administration as a co-formulation oradministration as separate compositions. As used herein, administrationin conjunction is meant to encompass any circumstance wherein an anti-Aβantibody and another agent are administered to an individual, which canoccur simultaneously and/or separately. As further enabled herein, it isunderstood that an anti-A Fab 2286-like antibody and optionally theother agent can be administered at different dosing frequencies orintervals. For example, an anti-Aβ antibody can be administered weekly,while the other agent can be administered less frequently. It isunderstood that the anti-Aβ antibody and the other agent can beadministered using the same route of administration or different routesof administration.

An “individual” (alternatively referred to as a “subject”) is a mammal,including a human. Other mammals include, but are not limited to, farmanimals (such as cows), sport animals, companion animals (such as cats,dogs, horses), primates, mice and rats.

The Fab 2286-like antibody may be formulated in a pharmaceuticalcomposition with a pharmaceutically acceptable carrier,“pharmaceutically acceptable carrier” includes any material which, whencombined with an active ingredient, allows the ingredient to retainbiological activity and is non-reactive with the subject's immunesystem. Examples include, but are not limited to, any of the standardpharmaceutical carriers such as a phosphate buffered saline solution,water, emulsions such as oil/water emulsion, and various types ofwetting agents. Diluents for aerosol or parenteral administrationinclude phosphate buffered saline or normal (0.9% /w/v) saline.Compositions comprising such carriers are formulated by well knownconventional methods (see, for example, Remington's PharmaceuticalSciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton,Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed.Mack Publishing, 2000).

The Fab 2286-like antibody may include additional modifications whichinclude functionally equivalent antibodies which do not significantlyaffect their properties and variants which have enhanced or decreasedactivity and/or affinity. For example, the amino acid sequence of theheavy or light variable region of the Fab 2286-like antibody may bemutated to obtain an antibody with a desired binding affinity to Aβ₁₋₄₂of Aβ₁₋₄₃ peptide. Modification of polypeptides is routine practice inthe art and need not be described in detail herein. Examples of modifiedpolypeptides include polypeptides with conservative substitutions ofamino acid residues, one or more deletions or additions of amino acidswhich do not significantly deleteriously change the functional activity,or use of chemical analogs.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto an epitope tag. Other insertional variants of the antibody moleculeinclude the fusion to the N- or C-terminus of the antibody of an enzymeor a polypeptide which increases the serum half-life of the antibody orwhich provides a particular functionality.

Substitution variants have at least one amino acid residue in theantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable regions, but Fc alterations are also contemplated.Conservative substitutions are shown in Table 3 under the heading of“conservative substitutions”.

TABLE 3 Amino Acid subiitutions Original Conservative ResidueSubstitutions Exemplary Substitutions Ala (A) Val Val; Leu; Ile Arg (R)Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp; Lys; Arg Asp (D) Glu Glu;Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; Gln Glu (E) Asp Asp; Gln Gly(G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met;Ala; Phe; Norleucine Leu (L) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys(K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val;Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W)Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met;Phe; Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining: (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation; (b) the charge or hydrophobicity of the moleculeat the target site; or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties: (1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile; (2)Polar without charge: Cys, Ser, Thr, Asn, Gin; (3) Acidic (negativelycharged): Asp, Glu; (4) Basic (positively charged): Lys, Arg; (5)Residues that influence chain orientation: Gly, Pro; and (6) Aromatic:Trp, Tyr, Phe, His. As taught herein, the Fab 2286-like antibodycomprises a substitution of E to an R in the sequence WIGE in the heavychain variable region of Fab 2286 (wild type).

Non-conservative substitutions are made by exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcross-linking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability, particularly where the antibody is an antibodyfragment such as an Fv fragment.

Amino acid modifications can range from changing or modifying one ormore amino acids to complete redesign of a region, such as the variableregion. Changes in the variable region can alter binding affinity and/orspecificity.

Modifications also include glycosylated and nonglycosylatedpolypeptides, as well as polypeptides with other post-translationalmodifications, such as, for example, glycosylation with differentsugars, acetylation, and phosphorylation. Antibodies are glycosylated atconserved positions in their constant regions (Jefferis and Lund (1997)Chem. Immunol. 65:111-128; Wright and Morrison (1997) TibTECH 15:26-32).The oligosaccharide side chains of the immunoglobulins affect theprotein's function (Boyd et al. (1996) Mol. Immunol. 32:1311-1318;Wittwe and Howard (1990) Biochem. 29:4175-4180) and the intramolecularinteraction between portions of the glycoprotein, which can affect theconformation and presented three-dimensional surface of the glycoprotein(Jefferis and Lund (1997) supra; Wyss and Wagner (1996) Current Opin.Biotech. 7:409-416). Oligosaccharides may also serve to target a givenglycoprotein to certain molecules based upon specific recognitionstructures. Glycosylation of antibodies has also been reported to affectantibody-dependent cellular cytotoxicity (ADCC).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

The glycosylation pattern of antibodies may also be altered withoutaltering the underlying nucleotide sequence. Glycosylation largelydepends on the host cell used to express the antibody. Since the celltype used for expression of recombinant glycoproteins, e.g. antibodies,as potential therapeutics is rarely the native cell, variations in theglycosylation pattern of the antibodies can be expected (see, e.g. Hseet al. (1997) J. Biol. Chem. 272:9062-9070).

The methods taught herein use antibodies (including pharmaceuticalcompositions comprising the antibodies) that specifically bind to an Aβpeptide at the C-terminal end. The antibodies are further characterizedby any (one or more) of the following characteristics: (a) suppressesformation of amyloid plaques in a subject; (b) reduces amyloid plaquesin a subject; (c) treats, prevents, ameliorates one or more symptoms ofAlzheimer's disease; (d) improves cognitive function. The antibodiesdescribed herein may exhibit a desirable safety profile, for example,the compositions of Fab 2286-like antibody do not cause significant orunacceptable levels or have a reduced level of any one or more of:bleeding in the brain vasculature (cerebral hemorrhage);meningoencephalitis (including changing magnetic resonance scan);elevated white blood count in cerebral spinal fluid; central nervoussystem inflammation.

The Fab 2286-like antibodies, polynucleotides encoding amino acid chainsof the Fab 2286-like antibody, and pharmaceutical compositions describedherein can be used in methods for treating, preventing and inhibitingthe development of a disease characterized by aberrant deposition of Aβpeptide in the brain of a subject. The methods comprise administering tothe subject an effective amount of the Fab 2286-like antibody thatspecifically binds to the Aβ peptide or the Aβ peptide deposit or apolynucleotide encoding a chain of the antibody.

Without limiting the present disclosure to any one hypothesis or mode ofaction, the Fab 2286-like antibody can act directly in the brain toinduce microglial-mediated removal of Aβ or via the “peripheral sink”route. According to the latter mode of action, the Fab 2286-likeantibody does not need to cross the blood-brain barrier to act, butrather, binds Aβ in the blood and shift the equilibrium of Aβ from theCNS to the plasma, where Aβ can be degraded (DeMattos et al. (2001) ProcNatl Acad Sci USA 98:8850-8855).

The Fab 2286-like antibodies, polynucleotides encoding an amino acidchain in the antibody, and pharmaceutical compositions described hereincan be used in methods for treating, preventing and inhibiting thedevelopment of Alzheimer's disease and other diseases associated withaltered Aβ or APP expression, or accumulation or deposit of Aβ peptide(collectively termed “Aβ-associated diseases”), such as Down's syndrome,Parkinson's disease, multi-infarct dementia, mild cognitive impairment,cerebral amyloid angiopathy, glaucoma, vascular disorder caused bydeposit of Aβ peptide in blood vessels (such as stroke and HCHWA-D).Such methods comprise administering the Fab 2286-like antibodies or apharmaceutical composition comprising same to the subject. Inprophylactic applications, pharmaceutical compositions or medicamentsare administered to a patient susceptible to, or otherwise at risk of,Alzheimer's disease (or other Aβ-associated disease) in an amountsufficient to eliminate or reduce the risk, lessen the severity, ordelay the outset of the disease, including biochemical, histologicaland/or behavioral symptoms of the disease, its complications andintermediate pathological phenotypes presenting during development ofthe disease. In therapeutic applications, compositions or medicamentsare administered; to a patient suspected of, or already suffering fromsuch a disease in amount sufficient to cure, or at least partiallyarrest, the symptoms of the disease (biochemical, histological and/orbehavioral), including its complications and intermediate pathologicalphenotypes in development of the disease.

The present disclosure teaches a method of delaying development of asymptom associated with Alzheimer's disease (or other Aβ-associateddisease) in a subject comprising administering an effective dosage of apharmaceutical composition comprising a Fab 2286-like antibody describedherein to the subject. Symptoms associated with Alzheimer diseaseincludes, but not limited to, abnormalities of memory, problem solving,language, calculation, visuospatial perception, judgment, and behavior.

This disclosure enables a method of inhibiting or suppressing theformation of amyloid plaques and/or Aβ accumulation in a subjectcomprising administering an effective dose of a pharmaceuticalcomposition comprising a Fab 2286-like antibody to the subject. In anembodiment, the amyloid plaques are in the brain of the subject. In anembodiment, the amyloid plaques are in the cerebral vasculature of thesubject. In an embodiment, the Aβ accumulation is in the circulatorysystem of the subject.

The instant disclosure teaches a method of reducing amyloid plaquesand/or reducing or slowing Aβ accumulation in a subject comprisingadministering an effective dose of a pharmaceutical compositioncomprising an Fab 2286-like antibody to the subject an Fab 2286-likeantibody. In an embodiment, the amyloid plaques are in the brain of thesubject. In an embodiment, the amyloid plaques are in the cerebralvasculature of the subject. In an embodiment, the Aβ accumulation is inthe circulatory system of the subject.

Further taught herein is a method of removing or clearing amyloidplaques and/or Aβ accumulation in a subject comprising administering aneffective dose of a pharmaceutical composition comprising an Fab2286-like antibody to the subject an Fab 2286-like antibody. In anembodiment, the amyloid plaques are in the brain of the subject. In someembodiments, the amyloid plaques are in the cerebral vasculature of thesubject. In an embodiment, the Aβ accumulation is in the circulatorysystem of the subject.

The present disclosure is instructional for a method of reducing Aβpeptide in a tissue (such as brain), inhibiting and/or reducingaccumulation of Aβ peptide in a tissue (such as brain), and inhibitingand/or reducing toxic effects of Aβ peptide in a tissue (such as brain)in a subject comprising administering an effective dose of apharmaceutical composition comprising an Fab 2286-like antibody to thesubject an Fab 2286-like antibody. Aβ polypeptide may be in soluble,oligomeric, or deposited form. An oligomeric form of Aβ may be composedof 2 or more (e.g. 2 to 50) Aβ polypeptides, which can be a mixture ofinter alia A β₁₋₄₀, Aβ₁₋₄₂ and A β₁₋₄₃ peptides as well as Aβ₃₋₄₂,Aβ₄₋₄₂, A β_(pyroGlu3-42) and A β_(pyroGlu11-42) and the like.

The present disclosure teaches a method of improving cognition orreversing cognitive decline associated with diseases associated withamyloid deposit of Aβ in a subject, such as Alzheimer's disease,comprising administering an effective dosage of a pharmaceuticalcomposition comprising an Fab 2286-like antibody to the subject an Fab2286-like antibody.

Effective dosages and schedules for administering the Fab 2286-likeantibody may be determined empirically, and making such determinationsis within the skill in the art. Those skilled in the art will understandthat the dosage of antibody that must be administered will varydepending on, for example, the mammal that will receive the antibody,the route of administration, the particular type of antibody used andother drugs being administered to the mammal. Guidance in selectingappropriate doses for antibody is found in the literature on therapeuticuses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone etal. eds. Noges Publications, Park Ridge, N.J. (1985) ch. 22:303-357;Smith et al. (1977) Antibodies in Human Diagnosis and Therapy, Haber etal. eds., Raven Press, New York:365-389. A typical daily dosage of theantibody used alone might range from about 1 μg/kg to up to 100 mg/kg ofbody weight or more per day, depending on the factors mentioned above.Generally, any of the following doses may be used: a dose of at leastabout 50 mg/kg body weight; at least about 10 mg/kg body weight; atleast about 3 mg/kg body weight; at least about 1 mg/kg body weight; atleast about 750 μg/kg body weight; at least about 500 μg/kg body weight;at least about 250 μg/kg body weight; at least about 100 μg/kg bodyweight; at least about 50 μg/kg body weight; at least about 10 μg/kgbody weight; at least about 1 μg/kg body weight, or more, isadministered. Antibodies may be administered at lower doses or lessfrequent at the beginning of the treatment to avoid potential sideeffect. Administration may be by any route including intracerebral andintravenous. The latter is useful in promoting a “peripheral sink”effect.

In an embodiment, more than one antibody is present. Such compositionsmay contain at least one, at least two, at least three, at least four,at least five different antibodies which bind to different epitopes onAβ or which bind to the same epitope but with different bindingavidities.

The Fab 2286-like antibody may also be administered to a mammaliansubject in combination with effective amounts of one or more othertherapeutic agents. The antibody may be administered sequentially orconcurrently with the one or more other therapeutic agents. The amountsof antibody and therapeutic agent depend, for example, on what type ofdrugs are used, the pathological condition being treated, and thescheduling and routes of administration but would generally be less thanif each were used individually.

Following administration of antibody to the mammal, the mammal'sphysiological condition can be monitored in various ways well known tothe skilled practitioner. In an embodiment, the mammal is a human,companion animal, simian animal or laboratory test animal such as amouse or rat.

The instant disclosure further teaches articles of manufacture and kitscontaining materials useful for treating pathological conditionsdescribed herein, such as Alzheimer's disease or other Aβ-associateddiseases (such as Down's syndrome, Parkinson's disease, multi-infarctdementia, mild cognitive impairment, cerebral amyloid angiopathy,glaucoma, vascular disorder caused by deposit of Aβ peptide in bloodvessels [such as stroke and HCHWA-D]), or detecting or purifying Aβ orAPP. The article of manufacture comprises a container with a label.Suitable containers include, for example, bottles, vials, and testtubes. The containers may be formed from a variety of materials such asglass or plastic. The container holds a composition having an activeagent which is effective for treating pathological conditions or fordetecting or purifying Aβ or APP. The active agent in the composition isan Fab 2286-like antibody and may further include an anti-Fab 2286-likeantibody labeled with a reporter molecule or enzyme. The label on thecontainer indicates that the composition is used for treatingpathological conditions such as Alzheimer's disease or detecting orpurifying Aβ or APP, and may also indicate directions for either in vivoor in vitro use.

The present disclosure also provides kits comprising the Fab 2286-likeantibody and/or polynucleotides encoding amino acid chains of Fab2286-like antibody. In an embodiment, the kit disclosed herein comprisesthe container described above. In an embodiment, the kit comprises thecontainer described above and a second container comprising a buffer. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for performing anymethods described herein (such as methods for treating Alzheimer'sdisease, and methods for inhibiting or reducing accumulation of Aβpeptide in the brain). In kits to be used for detecting or purifying Aβor APP, the antibody is typically labeled with a detectable marker, suchas, for example, a radioisotope, fluorescent compound, bioluminescentcompound, a chemiluminescent compound, metal chelator or enzyme.

In an embodiment, described herein is a composition for use in any ofthe methods described herein, whether in the context of use as amedicament and/or use for manufacture of a medicament. Hence, taughtherein is a use of an antibody which binds to Aβ₁₋₄₀ or C-terminallyelongated forms thereof or covalently linked multimers thereof, theantibody comprising a modified Fab 2286 wherein a mature heavy chaincomprises a modified amino acid sequence set forth in SEQ ID NO: 1 withthe proviso that the amino acid residue at position 50 is not glutamicacid, wherein the numbering of the amino acid sequence WIGE in Fab 2286(SEQ ID NO:1) represents amino acid residues 47 to 50 or comprising oneor other more amino acid substitutions, additions and/or deletions tothe amino acid sequence of SEQ ID NO:2 in the manufacture of amedicament for the treatment of pathogenic Aβ disease. In an embodiment,the amino acid is selected from the list consisting of alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, valine, pyrrolysineand selenocysteine. In an embodiment, the amino acid is a basic aminoacid residue such as arginine, lysine or histidine. In an embodiment,the amino acid is alanine. In a particular embodiment, the amino acid isarginine. In an embodiment, the Aβ disease is selected from the listconsisting of Alzheimer's disease, Down's syndrome, cognitive impairmentor memory loss, Parkinson's disease, multi-infarct dementia, cerebralamyloid angiopathy, glaucoma and a vascular disorder caused bypathogenic Aβ peptide in blood vessels (e.g. stroke and hereditarycerebral hemorrhage with amyloidosis-Dutch type [HCHWA-D]).

Another aspect enabled herein is a method of detecting a toxic form ofAβ in a sample from a subject, said method comprising identifyingbinding between the Aβ form and Fab 2286-like antibody.

Diagnostic applications include the detection of Alzheimer's disease,Down's syndrome, cognitive impairment or memory loss, Parkinson'sdisease, multi-infarct dementia, cerebral amyloid angiopathy, glaucomaand a vascular disorder caused by pathogenic Aβ peptide in blood vessels(e.g. stroke and hereditary cerebral hemorrhage with amyloidosis-Dutchtype [HCHWA-D]) as well as pre-eclampsia.

In an embodiment, the Fab 2286-like antibody is used to detectAlzheimer's disease, HCHWA-D or pre-eclampsia.

Any immunoassay may be used to capture and/or directly identify a toxicform of Aβ. Immunoassays are binding assays. Certain immunoassayscontemplated herein are the various types of enzyme linked immunosorbentassays (ELISAs) and radioimmunoassays (RIA) known in the art. However,it will be readily appreciated that detection is not limited to suchtechniques, and Western blotting, dot blotting, FACS analyses, and thelike may also be used.

In an embodiment, the assay is capable of generating quantitativeresults.

The Fab 2286-like antibody is either labeled with a reporter molecule orthe Fab 2286-like antibody is used to bind to or capture the Aβ and asecond antibody (directed to Fab 2286-like antibody or a portion of Aβ)labeled with a reporter molecule is used to detect an Fab 2286-likeantibody-Aβ complex.

The Fab 2286-like antibody can be used to screen for or capture toxicforms of Aβ. Techniques for the assays contemplated herein are known inthe art and include, for example, sandwich assays and ELISA.

It is within the scope of this invention to include any secondantibodies (monoclonal, polyclonal or fragments of antibodies orsynthetic antibodies) directed to Fab 2286-like antibody. Both types ofantibodies may be used in detection assays or the Fab 2286-like antibodymay be used with a commercially available anti-immunoglobulin antibody.

Both polyclonal and monoclonal antibodies specific for Fab 2286-likeantibody are obtainable by immunization with Fab 2286-like antibodies orantigenic fragments thereof and either type is utilizable forimmunoassays. The methods of obtaining both types of sera are well knownin the art. Polyclonal sera are less preferred but are relatively easilyprepared by injection of a suitable laboratory animal with an effectiveamount of the Fab 2286-like antibody, or antigenic parts thereof,collecting serum from the animal and isolating specific sera by any ofthe known immunoadsorbent techniques. Although antibodies produced bythis method are utilizable in virtually any type of immunoassay, theyare generally less favoured because of the potential heterogeneity ofthe product.

The use of monoclonal antibodies in an immunoassay is particularlypreferred because of the ability to produce them in large quantities andthe homogeneity of the product. The preparation of hybridoma cell linesfor monoclonal antibody production derived by fusing an immortal cellline and lymphocytes sensitized against the immunogenic preparation canbe done by techniques which are well known to those who are skilled inthe art.

Another aspect of the present invention contemplates, therefore, amethod for detecting a toxic form of Aβ in a biological sample from asubject. The method comprising contacting the biological sample with Fab2286-like antibody for a time and under conditions sufficient for anantibody-polypeptide complex to form, and then detecting the complex.The Fab 2286-like antibody may be labeled with a reporter molecule andthis is detected or a labeled anti-Fab 2286-like antibody or genericlabeled anti-immunoglobulin antibody is employed to detect the Fab2286-like antibody-A complex.

A biological sample includes a blood or cerebral spinal fluid sample.For the detection of pre-eclampsia, the sample is urine.

A “labeled” antibody means an antibody labeled with a reporter molecule.By “reporter molecule” as used in the present specification, is meant amolecule which, by its chemical nature, provides an analyticallyidentifiable signal which allows the detection of antigen-boundantibody. Detection may be either qualitative or quantitative. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide containing molecules (i.e.radioisotopes) and chemiluminescent molecules. In the case of an enzymeimmunoassay, an enzyme is conjugated to a second antibody, generally bymeans of glutaraldehyde or periodate. As will be readily recognized,however, a wide variety of different conjugation techniques exist, whichare readily available to the skilled artisan.

Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labeled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic colour visually detectable with a lightmicroscope. The fluorescent labeled antibody is allowed to bind to a Fab2286-like antibody-Aβ complex. After washing off the unbound reagent,the remaining tertiary complex is then exposed to the light of theappropriate wavelength the fluorescence observed indicates the presenceof the hapten of interest. Immunofluorescene and EIA techniques are bothvery well established in the art. However, other reporter molecules,such as radioisotope, chemiluminescent or bioluminescent molecules, mayalso be employed. In an embodiment, taught herein is a dipstickcomprising Fab 2286-like antibody immobilized thereon for use indetecting toxic forms of Aβ in a biological sample. In an embodiment,the sample is urine and the condition diagnosed is pre-eclampsia.

EXAMPLES

Aspects of the present disclosure are now further described by thefollowing non-limiting Examples.

Materials and Methods Antibody Cloning and Expression and Purification

A chimeric antibody was produced incorporating light and heavy chainvariable domains of the anti-Aβ murine monoclonal antibody, Mab 2286,described by Rosenthal et al. (US Patent Application Nos. 2004/046512and 2007/0160616) conjugated, respectively, to human kappa 1immunoglobulin (hKappa) light chain and gamma-1 immunoglobulin (hG1)heavy chain constant regions. Amino acid sequences for the Fab region ofthe light (V_(L)+C_(L)) [SEQ ID NO:3] and heavy (V_(H)+C_(H)) [SEQ IDNO: 1] chains of Fab 2286 are shown in Table 4.

Heavy and light chain constructs were each cloned into the pcDNA 3.1vector (Invitrogen). Plasmids were transformed into E. coli (DHS-alpha,Invitrogen) for amplification under ampicillin selection and purifiedwith a PureLink (Trade Mark) HiPure Plasmid Megaprep Kit (Invitrogen)according to the manufacturer's instructions. Recombinant expressionplasmids were then transfected into FreeStyle (Trade Mark) 293-F cellsto allow expression of the recombinant chimeric antibody.

FreeStyle (Trade Mark) 293-F cells were cultured in FreeStyle expressionmedium (Invitrogen), and maintained at 37° C. with an atmosphere of 8%v/v CO₂. Transient transfections were performed using 293Fectintransfection reagent (Invitrogen) according to the manufacturer'sinstructions. Cultures of 1000 mL were shaken in a Cellbag (Trade Mark)2L (flow rate: 0.1-0.15 Lpm) on a bioreactor system. Cultures weresupplemented with 0.1% Pluronic F68 (Invitrogen) and 0.5% LucratoneLupin (Millipore) 4 and 24 hours post transfection, respectively, andthe rocking angle adjusted to 9° after 24 hours. Six dayspost-transfection the cell culture supernatants were harvested bycentrifugation at 1000×g.

Harvested conditioned media was filtered through a 0.22 μm filter andapplied to a MabSelect SuRe HiTrap Protein A HP Column (5 ml, Hitrap, GELife Sciences, Sweden) previously equilibrated with PBS. Antibody waseluted with 0.1 M sodium citrate pH 3.5 and collected into 10% v/v finalvolume of 3.0 M Tris pH 8.0. Antibody was buffer exchanged into PBSusing a HiLoad desalting column 26/10 (GE Life Sciences, Sweden) andconcentrated to 1 mg/mL with a centrifugal concentrator (Amicon ultra,50 kDa MWCO) for storage at −80° C.

For expression and purification of Fab2286-like antibody fragment,synthetic DNA cloned into pcDNA3.1 expression vectors were obtained fromGenscript for expression of the heavy and light chains. The N-terminalsignal peptides were incorporated for the heavy chain (MGWSWIFLFLVSGTGGVLSE) and light chain (MESQTQVLMS LLFWVSGTCG). The heavy chain wasexpressed as a Hexa-histidine tagged construct, with the tag at theC-terminus of the chain. DNA constructs were transformed into DHSα E.coli for amplification under ampicillin selection and purified with aPureLink (Trade Mark) HiPure Plasmid Megaprep Kit (Invitrogen) accordingto the manufacturer's instructions. Recombinant expression plasmids werethen co-transfected into FreeStyle (Trade Mark) 293-F cells (Invitrogen)to allow expression of the recombinant antibody fragment.

293-F cells were cultured in FreeStyle expression medium (Invitrogen),and maintained at 310 K with an atmosphere of 8% v/v CO₂. The expressionwas performed in 4 L batches in a Certomat Ct plus incubator (Sartorius)by doing co-transfection of 1×10⁶ cells/mL with both DNA and 293Fectintransfection reagent (Invitrogen) according to the manufacturer'sinstructions. Cultures were supplemented with 5 mL/L of 10% v/v PluronicF68 (Invitrogen), 5 mg/L Lucratone Lupin (Millipore) 4 hours posttransfection, and 5 mg/L of glucose 2 days post-transfection. The cellculture supernatants were harvested by centrifugation at 500×g, and themedia collected for purification.

4 L of harvested media was concentrated to 200 mL by tangental flowfiltration system (Millipore Proflux M12). The concentrated media wascentrifuged at 20000×g for 30 minutes before being purified byimmobilised-metal affinity chromatography. The supernatant containingFab was incubated for 1 hour with equilibrated Ni-NTA affinity resin(Qiagen). The mixture was washed 4 times with 20 mM Tris pH 8.0, 150 mMNaCl and 20 mM imidazole. The protein of interest was eluted with 20 mMTris pH 8.0, 150 mM NaCl and 500 mM imidazole. Eluted sample was furtherpurified by size exclusion chromatography with a HiLoad Superdex 20020/60 run in PBS on an AKTApurifier (GE Healthcare). Fractions wereconcentrated to 2 mg/mL with a centrifugal concentrator (Amicon ultra,10 kDa MWCO).

Purification of Fab

Fab fragments were prepared using the Immunopure Fab Preparation kit(Pierce Biotechnology). IgG (10 mg) was digested with 0.5 mL of the 50%w/v immobilized papain slurry overnight at room temperature. Theimmobilized papain was removed by centrifugation (3000×g for 3 minutes)and the supernatant containing the digested sample was loaded onto a 1mL HiTrap Protein A column (GE Life Sciences, Sweden). Fractionscontaining the clean Fab fragments were pooled and dialyzed over nightin 20 mM HEPES pH 7.0. The dialyzed sample was concentrated with aCentriprep-10 centrifugal concentrator to 4 mg/mL (10 kDa MWCO,Millipore). The digestion and purification steps were monitored bySDS-PAGE.

Amyloid-β Peptides

Peptides corresponding to residues 1-28 (Aβ28), 1-40 (Aβ40) and 1-42(Aβ42) of the amyloid-β sequence (1-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA-42) [SEQ ID NO:6] were obtained from commercial sources:Aβ28 (AnaSpec, San Jose, Calif., USA); Aβ40 and Aβ42 (GenicBio BioTechCo., Shanghai, China). Each of the Aβ peptides was resuspended in 100%(v/v) 2,2,2-trifluoroethanol (TFE) and aliquoted to give 100 μg perEppendorf tube. All aliquots were freeze-dried for 4 hours and stored at−80° C. until required.

SDS-PAGE and Western Blot

Aβ peptides (100 μg) were dissolved in 2 μL of 10 μM NaOH, then made upto a volume of 100 μL with PBS. Samples of Aβ40 and Aβ42 were preparedin advance and aged at room temperature for either 24 hours or 21 days.A 1:1 molar ratio of Aβ40 and Aβ42 was also prepared and allowed to ageovernight before running on SDS-PAGE. Aβ28 was used as a negativecontrol and prepared on the day along with fresh samples of Aβ40 andAβ42.

An aliquot of 0.5 μg of all aged and freshly prepared Aβ peptides innon-reducing Lamelii loading dye was run on 12% w/v SDS polyacrilamidegel (NuPage; Invitrogen). Gels were run in triplicate, one gel wasCoomassie stained with InstantBlue (Expedon) and the other two weretransferred for 1 hour at 4° C. to nitrocellulose membrane (Millipore).

Following Western transfer, the nitrocellulose membrane was blocked with3% w/v BSA in PBS with 0.1% v/v Tween-20 (PBS-T) overnight at 4° C. Themembrane was incubated with either Mab 2286 (1:5000) or Mab WO2 (1:2000)in 3% v/v BSA, PBS-T for 1 hour at room temperature. It was then washedfour times in PBS-T for 15 minutes each. The blot was then incubatedwith 1:5000 Protein A-HRP (Millipore) in PBS-T for 1 hour at roomtemperature. It was washed a further four times in PBS-T for 15 minutes.The membrane was incubated with SuperSignal West Pico chemiluminescent(Pierce) for 5 minutes, exposed for 1 minute to X-ray film (Super RX;Fuji) and developed.

Crystallization

Initial crystallization screening resulted in small needles of crystalsthat were subsequently optimized by a combination of the Additive Screen(Hampton Research) and grid screening varying pH and PEG concentration.The best crystals were grown in 100 mM citric acid pH 5.5, 20% v/v2-propanol and 20% w/v PEG 4000. All crystals were grown using by vapordiffusion using 2 μL hanging drops at 22° C. Crystals were soaked in acryoprotectant (mother liquor plus 15% v/v glycerol) for several minutesprior to mounting in a N₂ stream at 100 K.

Structure Determination

Diffraction data from a single crystal of Fab 2286 were collected on theMX1 beamline of the Australian Synchrotron (Melbourne, Australia) withan ADSC Quantum 210r detector. Data collection were controlled usingBlue-Ice software (McPhillips et al. (2002) J. Synchroton Radiat9:401-406). All diffraction data were acquired from one crystal frozenat 100 K. Data collection statistics are summarized in Table 5.

Accession Numbers

The coordinates for the Fab 2286 structure have been deposited in theProtein Data Bank under the accession number 3U0W.

TABLE 4 Amino acid sequences of the chimeric Fab 2286light and heavy chains Light Chain (hKappa)DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYRKLPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 3) Heavy Chain (hG1)EVKLLESGGGLVQPGGSLKISCAASGFDFSRYWMNWVRQAPGKGLEWIGEINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTAIYYCARQMGYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 10)

Example 1 Western Blot Analysis

Antibody recognition of Aβ peptides is confounded by not only the rangeof different length peptides produced, but by their propensity to form arange of higher order complexes. In order to explore the specificity ofMab 2286, a number of different Aβ peptide preparations were examined.These included denatured and freshly dissolved Aβ28, Aβ40 and Aβ42,which would be mostly monomeric in solution. The peptides were allowedto oligomerize/aggregate both in isolation and as a mixture of the twomost common peptides, Aβ40 and Aβ42, for 24 hours to 21 days. These werethen analyzed by SDS-PAGE and detected by Coomassie stain (FIG. 1A),Western Blot with chimeric anti-Aβ Mab 2286 as the primary antibody(FIG. 1B) and Western Blot with murine anti-Aβ Mab WO2 as the primaryantibody (FIG. 1C).

Mab WO2 was used as the reference antibody as the 3-D Aβ recognitionepitope was previously determined, which involves the free side chainsof Aβ₂₋₇ (Miles et al. (2008) J Mol Biol 377:181-192). Consistent withthe epitope recognition of Mab WO2, specific bands corresponding toAβ28, Aβ40 and Aβ42 were detected as well as a range of higher molecularweight Aβ species observed for all of the aged peptides (FIG. 1C),indicating that the aging protocol used was sufficient to generate anumber of oligomeric forms.

By contrast, quite strikingly, Mab 2286 was highly selective andsensitive in detecting monomeric Aβ40, with no cross-reactivity with anyof the other aggregated forms apart from a minor Aβ40 dimeric species(FIG. 1B, Lane 2). There was no cross-reactivity with either the Aβ28 orAβ42 sample (FIG. 1B, Lane 6 and 4 respectively). Interestingly, nomonomeric Aβ40 was detected in the sample that had been left to age for21 days (FIG. 1B Lane 1), suggesting that all monomeric Aβ40 had reactedto form oligomers/aggregates. The preparation comprised a mixture ofAβ40 and Aβ42 and was allowed to age overnight. Under these conditionsthe level of monomeric Aβ40 detected by Mab 2286 was significantlyreduced (FIG. 1B Lane 5) relative to freshly prepared Aβ40 alone (thestarting concentration of monomeric Aβ40 is the same as the sample inLane 2) indicating that it has formed higher molecular weight oligomersin the presence of Aβ42.

Rosenthal and co-workers showed that Mab 2286 is specific for Aβterminating at residue 40. They also showed that single point alaninemutations across the terminal residues (35-MVGGVV-40 [SEQ ID NO:8])modulated binding affinity (Rosenthal et al. US Patent Application No.2007/0160616). Unlike the N-terminal region of Aβ, the C-terminal regionis hydrophobic. When combined, these data indicate that Mab 2286recognises the hydrophobic side chains of Aβ₄₀ at the C-terminus thatare predominantly available in the monomeric state or available forrecruitment from perhaps dimers or higher order complexes.

Example 2 Structure of Fab 2286

High resolution (2 Å) structure of a chimeric form of an antigen bindingfragment of Mab 2286 [Fab 2286], was determined (Table 5). The structureof Fab 2286 reveals that it is a typical immunoglobulin Fab heavy/lightchain heterodimer with an elbow angle of 185.2°. The putative Aβ bindinggroove in the Fab2286 crystal structure was calculated to beapproximately 1900{acute over (Å)}³ using Fred Receptor (v2.2.5, OpenEyeScientific Software, Inc., http://www.eyesopen.com).

TABLE 5 Data collection and refinement statistics Crystal Fab 2286 Datacollection Space group P2₁2₁2₁ Unit-cell parameters a (Å) 64.0 b (Å)81.3 c (Å) 108.5 Maximum resolution (Å) 2.0 Total observations 437605Unique reflections used 39062 Redundancy 11.7 (5.0)^([a]) Completeness(%) 95.7 (71.7) I/σ_(I) 27.9 (2.3) R_(SYM) (%)^([b]) 9.7 (83.1) Finalrefinement statistics Resolution (Å) 2.0 Total no. of atoms 3511 No. ofwaters 304 R-factor (%)^([c]) 18.9 R-free (%)^([d]) 23.5 r.m.s.d. bondlengths (Å) 0.021 r.m.s.d. bond angles (°) 2.0 Mean B-factor Proteinm.c. (s.c.) 28.4 (33.1) Solvent 41.2 Ramathandran regions (%) Favored90.0 Allowed 9.7 Disallowed 0.3 ^([a])The values in parentheses are forthe highest resolution bin. ^([b])R_(SYM) = Σ_(hkl)Σ_(i)|I_(i) −<I>|/|<I>|, where Ii is the intensity for the ith measurement of anequivalent reflection with indices h, k, l. ^([c])R-factor = Σ||F_(obs)|− |Fc_(alc)||/Σ|F_(obs)|, where F_(obs) and F_(calc) are the observedand calculated structure factor amplitudes respectively. ^([d])R-freewas calculated with 5% of the diffraction data that were selectedrandomly and not used throughout refinement.

Example 3 Generation of Fab 2286-Like Antibody

Mab 2286 exhibits specificity for the carboxyl terminus of Aβ₄₀ andshows no significant cross reactivity with C-terminally extendedpeptides such as Aβ₄₂ and Aβ₄₃. Antibody binding to Aβ₄₀ is notinhibited by Aβ₃₈ in competitive binding assays, and binding issimilarly modulated by conservative alanine single point mutationsacross the mapped residues (35-MVGGVV-40 [SEQ ID NO:8]).

The structure of chimeric Fab 2286 determined herein reveals a mechanismexplaining specificity for the Aβ₄₀ C-terminus and involves extensivecontacts with the Aβ₄₀ Val39 side chain buried in a hydrophobic pocketat the antibody interface. With the side chain of Aβ₄₀ Val39 anchored inthis way, the side chain of Val40 makes further hydrophobic contactssuch that the free carboxyl moiety can hydrogen bond with Asn35(H).C-terminally extended Aβ peptides would lose this potential hydrogenbond and the additional residues would force the ligand backbone toadopt a conformation that is incompatible with the Aβ binding site.

The Western Blot analysis shown in FIG. 1 indicates that, unlike theN-terminus, this hydrophobic C-terminal epitope is buried inoligomeric/aggregated synthetic Aβ₄₀, as only bands correlating withmonomer and dimer molecular weights are detected by the binding fragmentof Mab 2286, i.e. Fab 2286. It is concluded that the epitope of Aβrecognized by Fab 2286 is buried in higher order oligomeric assembliesis consistent with the crystal structures of the peptide reported byColletier et al. (2011) Proc Natl Acad Sci USA 108(41):16938-43. In thatwork, fibril-like structures of the peptide were shown to exhibitparallel and anti-parallel β-sheet stacked forms. The two steric zippersreported identify “knobs-in-holes” type packing, with Val39 (and Ile41)constituting the buried ‘knobs’ accommodated by the “holes” enabled bythe presence of the glycine residues.

It is known that amyloid plaques in the Alzheimer's diseased brain aremainly composed of C-terminally extended species such as Aβ₄₂ and Aβ₄₃,and these represent significant pathogens in Alzheimer's disease.According to the “peripheral sink” hypothesis (DeMattos et al. (2001)supra), anti-Aβ antibodies need not cross the blood brain barrier toact, but rather, bind Aβ in the blood and shift the equilibrium of Aβfrom the CNS to the plasma, where Aβ can be degraded.

A His-tagged fusion of Fab 2286 chains was generated using human kappa-1immunoglobulin (hkappa-1) light chain and gamma-1 immunoglobulin (hG1)heavy chain constant regions. In the Fab 2286, Glu50 is mutated to Arg(Glu50Arg) on the heavy chain. The Fab 2286-like antibody producedrecognized the hydrophobic C-terminal epitope available in monomericforms of Aβ, but one which can also accommodate C-terminally extended Aβpeptides which have a greater propensity to aggregate ornucleate/accelerate oligomerization.

Synthetic DNA constructs corresponding to heavy and light chains of Fab2286 were ordered from Genscript and subcloned into the pcDNA 3.1 vector(Invitrogen) for expression in mammalian cells. A single point mutationwas introduced into the heavy chain protein sequence based on acomputational analysis of Fab 2286, an antibody fragment that has onlylow micromolar affinity for amyloid beta peptide terminating at residue40. The mutant Fab was designed to recognize Aβ₁₋₄₀. It also was able tobind toxic C-terminally extended peptides including Aβ₁₋₄₂ and Aβ₁₋₄₃.The resultant mutant Fab not only bound the longer length peptides butalso did so with low nanomolar affinity. FIGS. 2 and 3 compare the Aβbinding profiles between Fab 2286 and the Fab 2286-like antibodyGlu50Arg Fab mutant against synthetic Aβ species and against Aβ speciesin brain tissue affected by familial and sporadic Alzheimer's diseaserespectively.

Example 4 Generation of Fab 2286-Like Antibody on a Murine Scaffold

A murinized form of the Fab 2286-like antibody was generated. Tomurinized the antibody, the following seuqences were employed:

Monoclonal antibody 2286 (Mab 2286) nucleic acid sequence: heavy chain[variable domain and constant domain 1 (CHI)]; [SEQ ID NO:12]:

gaggtgaagcttctcgagtctggaggtggcctggtgcagcctggaggatccctgaaactctcctgtgcagcctcaggattcgattttagtagatactggatgaattgggtccggcaggctccagggaaagggctagaatggattggagaaattaatccagatagcagtacgataaactatacgccatctctaaaggataaattcatcatctccagagacaacgccaaaaatacgctgtacctgcaaatgagcaaagtgagatctgaggaeacagccctttattactgtgcaagacaaatgggctactggggccaaggcaccactctcacagtctcctcagccaaaacgacacccccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgcctggtcaagggctatttccctgagccagtgacagtgacctggaactctggatccctgtccagcggtgtgcacaccttcccagctgtcctgcagtctgacctctacactctgagcagctcagtgactgtcccctccagcacctggcccagcgagaccgtcacctgcaacgttgcccacccggccagcagcaccaaggtggacaagaaaattgtgcccagggattgtand the light chain; (SEQ ID NO: 14):

gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagtgcaagtcagggcattagcaattatttaaactggtttcagcagaaaccagatggaactgttaaactcctgatctattacacatcaagtttacactcaggagtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagcaacctggaacctgaagatattgccacttactattgtcagcagtataggaagcttccgtacacgttcggaggggggaccaagctggaaataaaacgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgtThe Mab 2286 amino acid sequence: heavy chain [variable domain andconstant domain 1 (CHI)]; [SEQ ID NO: 13]:

EVKLLESGGGLVQPGGSLKLSCAASGEDFSRYWMNWVRQAPGKGLEWIGEINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCARQMGYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHP ASSTKVDKKIVPRDCand the light chain; (SEQ ID NO: 15):

DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYRKLPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC

The Fc portion from a murine gamma heavy chain (GenBank: AAA75163.1) wasadded to the heavy chain Fab portion sequence. The synthetic DNAconstruct was:

Gene name: MurineHeavyA_OPT, Length: 1391 bp. Sequence (SEQ ID NO: 16):

GAGGTCAAACTGCTGGAGAGTGGAGGGGGACTGGTGCAGCCAGGCGGGTCACTGAAGCTGAGCTGCGCCGCTTCCGGCTTCGACTTTTCCCGGTACTGGATGAATTGGGTGAGACAGGCTCCCGGAAAAGGCCTGGAGTGGATCGGGGAAATTAATCCTGATAGCTCCACCATCAACTACACACCAAGTCTGAAGGACAAATTCATCATTTCACGCGATAACGCAAAGAATACTCTGTATCTGCAGATGTCTAAAGTGCGAAGTGAGGACACCGCACTGTACTATTGTGCAAGACAGATGGGATACTGGGGACAGGGAACCACACTGACCGTGTCTAGTGCTAAGACTACCCCTCCCAGCGTGTATCCTCTGGCACCTGGCTCCGCAGCACAGACCAATTCTATGGTGACACTGGGCTGTCTGGTCAAGGGGTACTTCCCTGAGCCAGTCACAGTGACTTGGAACAGCGGCAGCCTGTCAAGCGGCGTGCACACCTTTCCTGCCGTCCTGCAGAGCGATCTGTATACACTGTCCTCTAGTGTCACTGTGCCCTCAAGCACCTGGCCTTCCGAGACCGTGACATGCAACGTCGCCCATCCTGCTTCCTCTACAAAGGTGGACAAGAAAATCGTCCCACGAGATTGCGGCTGTAAACCATGCATTTGTACTGTCCCCGAAGTGAGTTCAGTCTTCATCTTTCCACCCAAGCCAAAAGACGTGCTGACTATTACCCTGACACCCAAGGTCACATGCGTGGTCGTGGATATCAGCAAAGACGATCCCGAGGTGCAGTTCTCCTGGTTTGTCGACGATGTCGAAGTGCACACAGCCCAGACTCAGCCCAGGGAGGAACAGTTCAATTCTACCTTTCGCTCTGTGAGTGAGCTGCCTATTATGCATCAGGACTGGCTGAATGGAAAGGAATTCAAATGCAGAGTGAACTCTGCTGCTTTCCCGCTCCTATCGAGAAGACTATTAGCAAGACCAAAGGCAGGCCTAAAGCCCCACAGGTGTACACAATCCCTCCACCCAAGGAACAGATGGCTAAGGATAAAGTGAGCCTGACATGTATGATCACTGACTTCTTTCCCGAGGATATTACCGTGGAATGGCAGTGGAACGGGCAGCCCGCAGAGAACTATAAGAATACACAGCCTATTATGGACACTGATGGATCATACTTCGTGTATAGCAAGCTGAACGTCCAGAAATCTAATTGGGAAGCCGGCAACACTTTTACCTGTAGTGTGCTGCATGAAGGACTGCATAACCATCATACTGAAAAGTCACTGTCTCATTC ACCAGGCAAAand the amino acid sequence (SEQ ID NO: 17):

EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMNWVRQAPGKGLEWIGEINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCARQMGYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKSVLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

The mutant Fab 2286-like antibody (with the Glu to Arg substitution) wasderived from mutagenisis of that sequence yielding:

Variant name: Murine Heavy B, Variant sequence (SEQ ID NO: 18):

GAGGTCAAACTGCTGGAGAGTGGAGGGGGACTGGTGCAGCCAGGCGGGTCACTGAAGCTGAGCTGCGCCGCTTCCGCTTCGACTTTTCCCGGTACTGGATGAATTGGGTGAGACAGGCTCCCGGAAAAGGCCTGGAGTGGATCGGGCGTATTAATCCTGATAGCTCCACCATCAACTACACACCAAGTCTGAAGGACAAATTCATCATTTCACGCGATAACGCAAAGAATACTCTGTATCTGCAGATGTCTAAAGTGCGAAGTGAGGACACCGCACTGTACTATTGTGCAAGACAGATGGGATACTGGGGACAGGGAACCACACTGACCGTGTCTAGTGCTAAGACTACCCCTCCCAGCGTGTATCCTCTGGCACCTGGCTCCGCAGCACAGACCAATTCTATGGTGACACTGGGCTGTCTGGTCAAGGGGTACTTCCCTGAGCCAGTCACAGTGACTTGGAACAGCGGCAGCCTGTCAAGCGGCGTGCACACCTTTCCTGCCGTCCTGCAGAGCGATCTGTATACACTGTCCTCTAGTGTCACTGTGCCCTCAAGCACCTGGCCTTCCGAGACCGTGACATGCAACGTCGCCCATCCTGCTTCCTCTACAAAGGTGGACAAGAAAATCGTCCCACGAGATTGCGGCTGTAAACCATGCATTTGTACTGTCCCCGAAGTGAGTTCAGTCTTCATCTTTCCACCCAAGCCAAAAGACGTGCTGACTATTACCTGACACCCAAGGTCACATGCGTGGTCGGGATATCAGCAAAGACGATCCCGAGGTGCAGTTCTCCTGGTTTGTCGACGATGTCGAAGTGCACACAGCCCAGACTCAGCCCAGGGAGGAACAGTTCAATTCTACCTTTCGCTCTGTGAGTGAGCTGCCTATTATGCATCAGGACTGGCTGAATGGAAAGGAATTCAAATGCAGAGTGAACTCTGCTGCATTTCCCGCTCCTATCGAGAAGACTATTAGCAAGACCAAAGGCAGGCCTAAAGCCCCACAGGTGTACACAATCCCTCCACCCAAGGAACAGATGGCTAAGGATAAAGTGAGCCTGACATGTATGATCACTGACTTCTTTCCCGAGGATATTACCGTGGAATGGCAGTGGAACGGGCAGCCCGCAGAGAACTATAAGAATACACAGCCTATTATGGACACTGATGGATCATACTTCGTGTATAGCAAGCTGAACGTCCAGAAATCTAATTGGGAAGCCGGCAACACTTTTACCTGTAGTGTGCTGCATGAAGGACTGCATAACCATCATACTGAAAAGTCACTGTCTCATTCAC CAGGCAAAand the amino acid sequence (SEQ ID NO:19):

EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMNWVRQAPGKGLEWIGRINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCARQMGYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

The synthetic DNA light chain construct is:

Murine Light_OPT, Length: 725 bp, Sequence (SEQ ID NO:20):

GATATTCAGATGACTCAGACTACTTCTTCCCTGTCTGCAAGTCTGGGGGACCGAGTGACAATCTCATGCAGCGCCTCCCAGGGAATTTCCAACTACCTGAATTGGTTCCAGCAGAAGCCTGATGGCACAGTGAAACTGCTGATCTACTATACTAGCTCCCTGCACAGTGGGGTCCCATCAAGATTTTCTGGAAGTGGCTCAGGGACCGACTATAGCCTGACAATCTCCAACCTGGAGCCAGAAGATATTGCCACTTACTATTGCCAGCAGTACCGGAAGCTGCCCTATCTTTCGGCGGGGGAACCAAGCTGGAGATCAAAAGAGCTGACCGCCGCTCCCACCGTGAGCATTTTTCCCCCTTCTAGTGAACAGCTGACCTCTGGCGGGGCAAGTGTGGTCTGTTTCCTGAACAACTTCTACCCTAAAGACATCAACGTGAAGTGGAAAATTGATGAAGCGAGAGGCAGAACGGCGTCCTGAATTCCTGGACCGACCAGGATAGCAAGGACTCCACATATTCTATGTCAAGCACCCTGACACTGACTAAAGATGAGTACGAACGCCATAATAGCTATACATGTGAAGCTACTCATAAGACCTCTACCTCTCCTATTGTGAAATCTTTTAACCGAAATGAATGTand the amino acid sequence (SEQ ID NO:21):

DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYRKLPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEA THKTSTSPIVKSFNRNEC

The murinized Fab 2286-like antibody is used in animal model studies.

Example 5 Animal Model

Mouse model protocols for the evaluation of Fab 2286-like antibody areestablished. Efficacy of the antibody is tested in Tg2576 mice whichover express Aβ₄₀ or other mice which over express elongated forms ofAβ. To test Fab 2286-lie antibody or a derivative thereof, APP/PSI miceare used. An anti-AD drug efficacy protocol such as by Kenche et al.(2013) Angewandle Chemie International Edition 52):3374-3378 is areliable template to formulate a study design. These mice generatesignificant quantities of both Aβ₄₀ and Aβ₄₂ in the brain and areamyloid positive at around 7-8 months, cognitively impaired at 9-10months. Mice are purchased at ˜3 months of age from The JacksonLaboratory, Bar Harbor, Me., USA, and are allowed 2 months foracclimatization at a housing facility. APP/PSI mice are randomlyassigned to different treatment groups, (e.g. 10 mg/kg Fab 2286-likeantibody, 10 mg/kg vehicle control). Behavioral assessment and tissueanalysis follow protocols established by Kenche et al. (2013) supra.Treatment begins at 5 months before amyloid deposition has begun inthree arms of 15 animals each (based on a Power statistical analysis fora significant outcome). Behavioral assessments begin at 7 months of ageby Y-maze and water platform techniques as described by Kenche et al.(2013) supra. Weekly dosing is administered by oral gavage until 10months of age when tissue is collected and subject toimmunohistochemical analysis and examined for differential Aβ₄₀/Aβ₄₂₂profiles by SELDI-TOF MS and other IP/MS methods.

Example 6 Detection of Pre-Eclanmpsia

Urine samples are obtained from healthy women, healthy pregnant womenand pregnant women diagnosed with pre-eclampsia. The levels and speciesof Aβ are determined in each group. In an alternative, cerebral spinalfluid is tested. However, a urine sample is far less invasive. Fab2286-like antibody is used to either capture the Aβ species which isthen detected using a labeled anti-immunoglobulin antibody or the Fab2286-like antibody is labeled itself. It is expected that pregnant womenwith pre-eclampsia will exhibit elevated levels of brain-derived toxicAβ species comprising C-terminally elongated forms.

The Fab 2286-like antibody may also be used in the development of adipstick or other similar approach to detect toxic Aβ in urine ofpregnant women.

Example 7 Generation of Modified Fab 2286 Heavy Chains

Using the techniques disclosed herein or known to the skilled artisan, arange of substitutions is made at amino acid residue 50 in the heavychain variable region of Fab 2286.

The amino acid sequence set forth in SEQ ID NO:2 comprises an argininein place of a glutamic acid at residue 50. This creates the amino acidsequence comprising WIGR.

Other substitutions are selected from WIGA, WIGR, WIGN, WIGD, WIGC,WIGQ, WJGG, WIGH, WIGI, WIGL, WIGK, WIGM, WIGF, WIGP, WIGS, WIGT, WIGW,WIGY, WIGV, WIGO and WIGU.

An antibody comprising any of these substitutions from glutamic acid arereadily tested as disclosed herein for the ability to bind to Aβ₁₋₄₀ orC-terminally elongated forms.

Those skilled in the art will appreciate that the disclosure describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the disclosurecontemplates all such variations and modifications. The disclosure alsoenables all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of the steps or features orcompositions or compounds.

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1. An isolated Fab 2286-like antibody which binds to Aβ₁₋₄₀ and aC-terminally elongated form thereof, said antibody comprising a modifiedFab 2286 wherein a mature heavy chain variable region comprises an aminoacid sequence as set forth in SEQ ID NO: 1 with the proviso that theamino acid residue at position 50 is not glutamic acid (E), wherein thenumbering of the amino acid sequence WIGE in Fab 2286 (SEQ ID NO:1)represents residues 47 to 50 or comprising one or more other amino acidsubstitutions, additions and/or deletions to the amino acid sequence ofSEQ ID NO:
 1. 2. The isolated Fab 2286-like antibody of claim 1 hereinthe amino acid at position 50 is selected from the list consisting ofalanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine,pyrrolysine and selenocysteine.
 3. The isolated Fab 2286-like antibodyof claim 1 wherein the amino acid at position 50 is a basic amino acidselected from arginine, lysine and histidine.
 4. The isolated Fab2886-like antibody of claim 1 wherein the amino acid at position 50 isalanine.
 5. The isolated Fab 2286-like antibody of claim 1 wherein theamino acid at position 50 is arginine.
 6. The isolated Fab 2286-likeantibody of claim 1 wherein the C-terminally elongated form of Aβ₁₋₄₀ isselected from Aβ₁₋₄₂, Aβ₁₋₄₃, Aβ₃₋₄₂, Aβ₄₋₄₂, Aβ_(pyroGlu3-42) andAβ_(pyroGlu11-42).
 7. The isolated Fab 2286-like antibody of claim 1conjugated to constant regions of light and heavy chains.
 8. Theisolated Fab 2286-like antibody of claim 7 comprising a heavy chainvariable region selected from SEQ ID NO:9 and
 11. 9. A mammalianizedform of the isolated Fab 2286-like antibody of claim
 1. 10. Themammalianized form of the isolated Fab 2286-like antibody of claim 9wherein the antibody is humanized.
 11. An isolated nucleic acid moleculeencoding an amino acid chain of the antibody of any one of claims 1 to10.
 12. The isolated nucleic acid molecule of claim 11 comprised withinan expression vector.
 13. An isolated cell comprising the nucleic acidmolecule of claim 11 or
 12. 14. A pharmaceutical composition comprisingthe antibody of any one of claims 1 to 10, together with one or morepharmaceutically acceptable carriers, diluents and/or excipients. 15.The isolated Fab 2286-like antibody of any one of claims 1 to 10 for usein the treatment of pathogenic Aβ disease.
 16. The isolated Fab2286-like antibody of claim 15 wherein the Aβ disease is associated withor characterized by pathogenic forms of Aβ selected from Aβ₁₋₄₂, Aβ₁₋₄₃,Aβ₃₋₄₂, Aβ₄₋₄₂, Aβ_(pyroGlu3-42) and Aβ_(pyroGlu11-42) in tissue orfluid.
 17. The isolated Fab 2286-like antibody of claim 16 wherein thetissue is brain tissue.
 18. The isolated Fab 2286-like antibody of claim16 wherein the fluid is circulatory blood fluid.
 19. The isolated Fab2286-like antibody of any one of claims 15 to 18 wherein the disease isselected from Alzheimer's disease, Down's syndrome, cognitive impairmentor memory loss, Parkinson's disease, multi-infarct dementia, cerebralamyloid angiopathy, glaucoma, and a vascular disorder caused bypathogenic Aβ peptide in blood vessels.
 20. The isolated Fab 2286-likeantibody of claim 19 wherein the vascular disorder is stroke orhereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D).21. The isolated Fab 2286-like antibody of claim 19 wherein the diseaseis Alzheimer's disease.
 22. A method of treating or amelioratingsymptoms of a neurological condition selected from Alzheimer's disease,Down's syndrome, cognitive impairment or memory loss, Parkinson'sdisease, multi-infarct dementia, cerebral amyloid angiopathy, glaucoma,stroke and HCHWA-D or other adverse event of the systemic vasculature ina subject, the method comprising administering to the subject, aneffective amount of an Fab 2286-like antibody comprising a mature heavychain variable region having the amino acid sequence set forth in SEQ IDNO:1 with the proviso that the amino acid residue at position 50 is notgluatimic acid (E) using a numbering system where the amino acidsequence WIGE of Fab 2286 (SEQ ID NO:1) is at positions 47 to 50 orhaving one or more other amino acid substitutions, additions and/ordeletions thereto.
 23. The method of claim 22 wherein the amino acid atposition 50 is selected from the list consisting of alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, valine, pyrrolysine and selenocysteine.24. The method of claim 22 wherein the amino acid at position 50 is abasic amino acid selected from arginine, lysine and histidine.
 25. Themethod of claim 22 wherein the amino acid at position 50 is alanine. 26.The method of claim 22 wherein the amino acid at position 50 isarginine.
 27. A method for the treatment or prophylaxis of Alzheimer'sdisease in a subject, said method comprising administering to thesubject an effective amount of the Fab 2286-like antibody of any one ofclaims 1 to
 10. 28. A method of delaying development of a symptomassociated with Alzheimer's disease in a subject comprisingadministering to the subject an effective dosage of a pharmaceuticalcomposition comprising an Fab 2286-like antibody of any one of claims 1to
 10. 29. A method of inhibiting or suppressing the formation ofamyloid plaques and/or Aβ accumulation in a subject comprisingadministering to the subject an effective dose of a pharmaceuticalcomposition comprising an Fab 2286-like antibody of any one of claims 1to
 10. 30. A method of reducing amyloid plaques and/or reducing orslowing Aβ accumulation in a subject comprising administering to thesubject an effective dose of a pharmaceutical composition comprising anFab 2286-like antibody of any one of claims 1 to
 10. 31. A method ofremoving or clearing amyloid plaques and/or Aβ accumulation in a subjectcomprising administering to the subject an effective dose of apharmaceutical composition comprising an Fab 2286-like antibody of anyone of claims 1 to
 10. 32. A method of reducing Aβ peptide in a tissue,inhibiting and/or reducing accumulation of Aβ peptide in a tissue, orinhibiting and/or reducing toxic effects of Aβ peptide in a tissue in asubject comprising administering to the subject an effective dose of apharmaceutical composition comprising an Fab 2286-like antibody of anyone of claims 1 to
 10. 33. The method of any one of claims 22 to 32wherein the subject is a human.
 34. The method of claim 33 wherein theantibody is intravenously or intracerebrally administered.
 35. A kitcomprising the antibody of any one of claims 1 to
 10. 36. The kit ofclaim 35 further comprising an antibody labeled with a reporter moleculeor enzyme specific to the Fab 2286-like antibody.
 37. Use of theantibody of any one of claims 1 to 10 in the manufacture of a medicamentin the treatment of pathogenic Aβ disease.
 38. Use of claim 37 whereinthe Aβ disease is selected from the list consisting of Alzheimer'sdisease, Down's syndrome, cognitive impairment or memory loss,Parkinson's disease, multi-infarct dementia, cerebral amyloidangiopathy, glaucoma, and a vascular disorder caused by pathogenic Aβpeptide in blood vessels.
 39. Use of claim 38 wherein the vasculardisorder is stroke or hereditary cerebral hemorrhage withamyloidosis-Dutch type (HCHWA-D).
 40. Use of claim 37 wherein the Aβdisease is Alzheimer's disease.
 41. Use of Fab 2286-like antibody of anyone of claims 1 to 10 in the treatment of pathogenic Aβ disease via aperipheral sink.
 42. Use of claim 41 wherein the Aβ disease is selectedfrom the list consisting of Alzheimer's disease, Down's syndrome,cognitive impairment or memory loss, Parkinson's disease, multi-infarctdementia, cerebral amyloid angiopathy, glaucoma, pre-eclampsia and avascular disorder caused by pathogenic Aβ peptide in blood vessels. 43.Use of claim 42 wherein the vascular disorder is stroke or hereditarycerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D).
 44. Use ofclaim 41 wherein the Aβ disease is Alzheimer's disease.
 45. A method ofdetecting a toxic form of Aβ in a sample from a subject, said methodcomprising identifying binding between the Aβ form and Fab 2286-likeantibody.
 46. The method of claim 45 wherein the Fab 2286-like antibodyis labeled with a reporter molecule.
 47. The method of claim 45 whereinthe Fab 2286-like antibody is identified by a labeledanti-immunoglobulin antibody.
 48. The method of claim 45 or 46 or 47 forthe detection of a disease selected from the list consisting ofAlzheimer's disease, Down's syndrome, cognitive impairment or memoryloss, Parkinson's disease, multi-infarct dementia, cerebral amyloidangiopathy, glaucoma, pre-eclampsia and a vascular disorder caused bypathogenic Aβ peptide in blood vessels.
 49. The method of claim 48 forthe detection of Alzheimer's disease.
 50. The method of claim 48 for thedetection of stroke or hereditary cerebral hemorrhage withamyloidosis-Dutch type (HCHWA-D).
 51. The method of claim 48 for thedetection of pre-eclampsia.
 52. The method of claim 51 wherein the toxicform of Aβ is detected in urine.
 53. A dipstick comprising Fab 2286-likeantibody immobilized thereon for use in detecting toxic forms of Aβ in abiological sample.
 54. The dipstick of claim 53 wherein the sample isurine.
 55. The dipstick of claim 54 in the diagnosis of pre-eclampsia.56. An isolated polynucleotide encoding the amino acid sequence setforth in SEQ ID NO:19 or an amino acid sequence having at least 90%similarity thereto after optimal alignment.
 57. The isolatedpolynucleotide of claim 56 as set forth in SEQ ID NO:18 or a sequencehaving at lest 90% identity thereto after optimal alignment or anucleotide sequence which hybridizes to the complement of SEQ ID NO:18under medium or high stringency conditions.
 58. The isolatedpolynucleotide of claim 52 comprising the nucleotide sequence of SEQ IDNO:18.